International Journal of Immunology and Microbiology

In-silico structural and phylogenetic analysis of human beta globin gene family

Wed, 07 Dec 2022 00:00:00 +0000

There are 5 members of the human beta globin gene family. They have blood transportation function. There are some diseases related to the mutation in these gene. the common disease related to mutation these genes are sickle cell anemia, thalassemia etc. The structural and phylogenetic analysis is performed by using some bioinformatics tools. The gene sequence is retrieved and run under some tools to perform the specific analysis. The analysis performed include are similarity with other organism, determination of conserved domains and motifs, multiple sequence alignment, generation of phylogenetic tree, identification of exons and introns and determination of restriction sites. And the tools used for these analyses are BLASTP, pfam, clustal omega, MEGA7, gene structure display tool and serial cloner respectively. This study came out with a result that the genes diverged due to duplication and then mutation. The gene contain mostly non coding portion but there are 3 exons and 2 introns. The length of 2 introns is far more than that of 3 exons. In future the techniques like CRISPR/cas gene editing tool will be used to treat the disease related the beta globin gene family.

Key words:human beta globin gene, bioinformatics study of HBB, genome wise study of HBB, Beta subunit, hemoglobin beta subunit.

Can Infectious Diseases Be Prevented?

Haris S. Ansari , Pranav Adithya Navath — Fri, 01 Oct 2021 00:00:00 +0000

A study of infectious disease is described.The infectious disease pandemic has focused an intense spotlight on respiratory precautions for healthcare workers managing patients with respiratory viral infections. The author is discussed about the infections and recommended precautions for its prevention

Aspects Concerning Development Of Vaccine And Vaccination

Liana Monica DEAC — Tue, 09 Nov 2021 00:00:00 +0000

Vaccines can prevent or ameliorate infectious diseases morbidity and mortality. Vaccines generate immunity across the body as a whole, but they can also provoke specific immune responses in specific bodily areas. Vaccination includes various ways of administering immunogenes. Edward Jenner is considered the founder of vaccinology. With his importnata studies it was figured out that when a sufficiently large percentage of a population has been vaccinated, herd immunity results. The revolution of genetic engineering toward the end of the 20th century has greatly impacted vaccine development. Understanding the nature and cause of disease provides a basis for preventive action and control as even to improve the life quality by using the vaccination in the world.

Thyroidism Effect on Alopecia Patients In Pakistan

Muhhamd Waqar Mazhar — Tue, 09 Nov 2021 00:00:00 +0000

TSH is hormone is secreted by the anterior lobe of the pituitary gland and stimulate the secretions of the thyroid gland. Its secretions are controlled by the TSH-R on the epithelial cells of the thyroid gland. It controls the production of the thyroxin hormone from thyroid gland that is involved in the production of heat and energy. But the excessive amount of this hormone leads to the hair fall in some of the peoples. Hyper thyroidism correlates with the human skin and hair structure and its function. In case of hyperthyroidism hair bulb cell proliferation increases and hence hair fall rate also increases. While in case of hypothyroidism the bulb cell proliferation reduces and so, hair fall increases. Samples from different regions of the south Punjab were taken that were analyzed by the special chemistry analyzer (minividas). The concentrations of the T4, T3, and TSH were measured. Some of the alopecia patients were recorded high level TSH. The patients were treated with thyroxin tablets that reduces TSH level. After that the patients are also treated with hair fin tab, folli one shampoo, wistin tab, and multivitamins. Now the patients were observed with microscope. The growth of hair follicles and hairs were recorded. Hence, thyroxin tab use to decrease TSH level in body for 1 month. Hair fin tab, folli one shampoo, wistin tab and multivitamins are best treatment methods for the growth of hairs in alopecia patients.

Study of the Association between Helicobacter Pylori Infection and Primary open angle Glaucoma in China

Jianwei Zhou, Liang Han, Xiaoqing Chen, Yu Li, Dianyi Wang — Wed, 24 Mar 2021 00:00:00 +0000

Objective: To assess the relationship between Helicobacter pylori (Hp) infection and primary open-angle glaucoma (POAG); and meantime, to explore the possible mechanism of POAG induced by Hp. Methods: 30 consecutive POAG patients, 30 primary angle-closure glaucoma (PACG) and cataract patients were recruited and divided into three groups according to different diseases. The sera and aqueous humor samples were collected and used to detect Hp-specific IgG antibody (Hp-Ab) with dot immunogold filtration assay (DIGFA). ¹⁴C-urea breath test (¹⁴C-UBT) was carried out to detect Hp infection of all participants. Results: The Hp-Ab positive rate respectively was 76.7% (23/30) and 66.7% in sera samples and aqueous humor samples for POAG group, which was significantly higher than the corresponding data of the other two groups (all P<0.05). In ¹⁴C-UBT, the Hp-Ab positive rate was 63.3% in POAG group and it was close to that of serological result detected by DIGFA (P>0.05). There were little numbers of positive ANA and ENA in the three groups and no meaning to make statistically analysis. Conclusions: There is positive association between Hp infection and POAG, and the autoimmune is suggested as one of the key mechanisms in our opinions.

Introduction

Glaucoma is one of the commonest causes for blindness in the world. Generally, glaucoma is divided into primary open-angle glaucoma (POAG) and primary angle-closure glaucoma (PACG).¹ As a leading causes for blindness, the study of POAG causes more and more attention.^2,3To our understand, POAG is a chronic optic neuropathy characterized by atrophy and increased cupping of optic disk. To date, many aspects of its pathogenesis remain unknown but some significant risk factors are advanced age, African origin, familial history of glaucoma and elevated intraocular pressure.^4,5

Helicobacter pylori (Hp) is a Gram-negative and microaerophilic bacterium which plays an important role in the development of various upper gastrointestinal diseases. With the development of studies, some researchers reported that Hp was also associated with some extragastric diseases, such as ischemic heart disease,⁶ iron-deficient anemia,⁷ diabetes mellitus,⁸ and so on. In 2001, Kountouras et al⁹ established a higher prevalence of Hp infection in the sera of patients with POAG in a Greek population, and suggested a possible causal link between Hp and glaucoma. Subsequently, this finding was evidenced by some scholars in their own studies^.10 But the significance of such an association remains uncertain because of the conflicting findings reported by various studies.^11-13 Aiming to such a discrepancy, further studies are necessary.¹⁴

In this study, we just do detect Hp-specific IgG antibodies (Hp-Ab) in the sera and aqueous humor of patients with different ocular diseases, including POAG, PACG and cataract, and attempt to further determine the relationship between Hp infection and POAG and to analyze the possible mechanism of POAG induced by Hp.

Abbreviations

ANA, antinuclear antibody; ENA, Extractable nuclear antigen; DIGFA, dot immunogold filtration assay; Hp, Helicobacter pylori; Hp-Ab, Hp-specific IgG antibodies; PACG, primary angle-closure glaucoma; POAG, primary open-angle glaucoma; ¹⁴C-UBT: ¹⁴C-urea breath test.

Subjectsand methods

Subjects

30 consecutive POAG patients were enrolled with the average age of 68±7.3 y (ranged from 47 to 78 y). The ratio of the male and the female was 11: 19. Meantime, 30 PACG patients and 30 cataract patients were also recruited, and who were matched by age and sex with the POAG patients. According to different diseases, the participants were divided into POAG, PACG and cataract groups, respectively. All of them were excluded from tumor, immunodeficiency, autoimmune and infectious diseases in clinic, and also had no antibiotics and other medicines related to immunopotentiator or immunosuppressive agents in the six months before the experiment. Written informed consents were obtained from all the participants. The study was approved by the local ethics committee.

Hp-Ab detection of sera samples

2 ml venous blood was collected from each of the participants. The serum was obtained after centrifugation and used to detect Hp-Ab with dot immunogold filtration assay (DIGFA) according to the manufacturer’s instruction of the reagent kit (MP Biomedicals Asia-Pacific Pte. Ltd., Singapore).

Hp-Ab detection of aqueous humor samples

About 50 μl aqueous humor sample was aspirated at the beginning of glaucoma surgery from the each of the patients in the three groups, respectively. Hp-Ab was assayed with DIGFA as same as the detection process of venous blood samples.

Detection of Hp infection with ¹⁴C-urea breath test

Referring to Tang’s report,¹⁵¹⁴C-urea breath test (¹⁴C-UBT) was carried out in POAG group with Hp detection instrument-YH04 (Yanghe Medical Equipment Co. Ltd., China).

Sera auto-antibodies detection

Serum antinuclear antibody (ANA) was detected with the indirect immunofluorescence assay by a commercialized ANA kit. Extractable nuclear antigen (ENA) was assayed with line immunoassay. All reagents were bought from Jiangsu HOB Biotech Group, China.

Statistic analysis

Using T-test and Chi-square test, all analyses were performed with SPSS 13.0 software. P value less than 0.05 were considered significant.

Results

3.1 Hp infection detection in sera and aqueous humor

Of the sera samples, there were 23 cases exhibited Hp-Ab-positive in POAG group, and the positive rate was 76.7% which was significantly higher than those of PACG and cataract group (43.3% and 36.6% respectively). In the aqueous humor samples, there were 18 patients with positive Hp-Ab in POAG group, and the positive rate was 66.7%. Compared to each data of the other two groups, the difference was statistically significant (Table 1). In POAG group, the mean positive rate of sera samples was similar to that of aqueous humor and no difference existed between them (P = 0.287).

Table 1. The serum and aqueous humor qualitative test results of the patients with glaucoma

Hp infection detection with ¹⁴C-UBTAH: aqueous humor; a: POAG group vs cataract group; b: POAG group vs PACG group; c: PACG group vs cataract group.

In ¹⁴C-UBT, there were 19 patients with Hp-Ab-positive, and the positive rate was 63.3%. Compared to the data detected with DIGFA, the difference was not significant (Table 2).

Table 2. Comparison of DIGFA and 14C-UBT for diagnosis of Hp infection in POAG group

ANA and ENA detection* represents comparison of the positive rate detected with the two methods.

There were 4, 2 and 1 patients with ANA-positive in POAG, PACG and cataract group, respectively. The positive ENA in POAG group were SSA, SSB and Ro-52, and the corresponding numbers were 2, 2 and 1. Only Ro-52 showedpositive in PACG group while there was no positive ENA in cataract group (Table 3).

Table 3. The results for sera ANA, ENA of the patients of each group

Discussion

In Greece, a very active research group led by J. Kountouras published several original contributions as well as the reviews concerning the connection between Hp infection and POAG.^14,16 In other counties, there were also several papers containing the similar arguments issued, such as India,¹⁷ Turkey,¹⁸ Korea¹⁹ and so on. In China, Hong et al²⁰ detected Hp infection and POAG through ¹³C-UBT, and also found the positive correlation between them. Since then, there was no relative article issued by Chinese could be found in PubMed and other well-known scientific database. In this study, referring to other researchers’ reports, we designed and carried out the experiments. In the results, we found that the positive rate of sera Hp-Ab was high to 76.7% in POAG patients, which was significantly higher than those of the other two groups. This finding was close to the data of the previous reports^2,21 and further verified that there was a positive relation between Hp infection and POAG.

In the present study, we also assayed Hp infection with ¹⁴C-UBT. Encouragingly, the positive rate of Hp infection was 63.3%, which was very close to 76.7% detected with DIGFA. This result further indicated the existence of the relation between Hp infection and POAG. However, Bagnis et al²² thought that the studies based on Hp serological assessment might be misleading, since serum antibodies were not the sensitive markers of active Hp infection; while ¹³C-UBT could clarify the actual prevalence of POAG among patients infected by Hp. In fact, there were still deficiencies for ¹³C- or ¹⁴C -UBT, because it was more suitable for the detection of gastrointestinal Hp infection, and to an extent, there were false-negatives in the test.²³ This probably was the just reason for what the positive rate in DIGFA was little higher than that in ¹⁴C-UBT in this study. As to the cresyl fast violet staining on the histology preparations of tissue samples of trabeculum and iris introduced by Zavos et al,²⁴ although it could provide the direct and strong evidence for Hp infection in the pathophysiology of POAG, the difficult harvest of the sample limited its application. Therefore, in our opinions, the serological assay is suitable to detect Hp infectionand used to assess the relationship between Hp prevalence and POAG.

Except for detecting sera Hp-Ab, we also detected Hp-Ab in the aqueous humor collected from the majority of participants. As the results shown, the positive rate of the POAG group was statistically higher than each of the other groups, respectively. This result was consistent with that of the serological assessment and again showed the positive relation between Hp infection and POAG. However, in another similar study, Deshpande et al¹⁷also found a statistically significant difference between the POAG patients and the controls in the concentration of serum Hp-Ab, but they did not find any significant correlations between the Hp concentrations of the aqueous humor of the different patient groups. This disagreement probably associated with the damage degree of blood-brain barrier (BBB), because the sera Hp-Ab could reach the trabeculum and iris under the condition of the BBB disruption.²⁵ According to the results of the present study, we supported the hypothesis related to POAG onset that Hp-Ab in circulation might get through the blood-aqueous humor barrier, further condensed in aqueous humor and finally induced or aggravated glaucomatous damage.²

As to the occurrence of POAG, we thought another autoimmune mechanism was most probable and should not be ignored: Hp infection initiated autoimmune response because of the common genetic components shared in Hp and human nerve tissue; and then, cell destruction which mediated by apoptosis direct caused glaucoma.²⁶ Just based on the theory, we designed and detected sera ANA and ENA of the POAG patients and the control participants, and hoped to find any evidences related to autoimmune. As a result, we found that the positive rate of every group was rather low and there was no difference between them. However, this seronegative result can’t deny the hypothesis of autoimmune mechanism in POAG; and the auto-antibodies specific to eyes, such as trabeculum and iris, were suggested to be detected in future study in our opinions.

Conclusion

The positive association between Hp infection and POAG not only using serum sample but also aqueous humor sample is found in this study. And further, through the experimental data, it is suggested that the autoimmune induced by Hp infection probably is the key mechanism for POAG onset, and Hp detection should be taken as a routinized index applied to the prevention and therapy of POAG in clinic. However, we can not sufficiently investigate the possible mechanism of POAG relates to Hp infection. Is it true that Hp infection only relative to POAG but not a causative factor for POAG?¹⁸ What are the initial mechanisms of Hp in POAG if the pathogen takes part in the onset of the disease? Such questions will be the study topics to the medical researchers worldwide in future.

Funding

This work is supported by the Research Fund for Lin He’s Academician Workstation of New Medicine and Clinical Translation in Jining Medical University(JYHL2018FMS08), and the Project of scientific research support fund for teachers of Jining Medical University (JYFC2018FKJ023).

Conflicts of interest

There is no any conflict of interest between all of the authors.

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Kountouras J.; Mylopoulos N.; Konstas A. G.; Zavos C.; Chatzopoulos D.; Boukla A. Graefe’s Arch Clin Exp Ophthalmo. 2003, l241, 884.
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COVID-19 Treatments and Vaccines: A year in Review

Joseph Murphy — Wed, 02 Jun 2021 00:00:00 +0000

The SARS-CoV-2 coronavirus (COVID-19) pandemic has precipitated an enormous collaborative global effort within the scientific and medical community in search of therapeutic and preventative solutions. The aim of this review is to collate the key developments regarding pharmacological treatments tested and vaccine candidates that have been approved to treat and arrest the spread of COVID-19.

Introduction

COVID-19 Transmission

The COVID-19 outbreak has caused one of the most widespread and significant public health crises in decades. It has become one of the leading causes of death internationally. The primary route of transmission from person-to-person is from airborne aerosol spread through close physical contact, particularly in enclosed, poorly ventilated areas.⁽¹⁾ Transmission through contaminated objects was originally considered a major transmission contributor; however, it is no longer considered a significant driver of the spread. Wearing masks has shown to be effective at preventing or curtailing viral transmission, especially when combined with other measures like social distancing and depopulation of indoor communal spaces.⁽²⁾

Mechanism of action:

The mechanism of action and entry into human physiology at a cellular level has been described previously.⁽³⁾Briefly, the virus binds and enters the host cell through a spike protein expressed on its surface. The infection begins when the long protruding spike proteins that latches on to the angiotensin-converting enzyme 2 (ACE-2), a receptor involved in regulating blood pressure ACE-2 protein. From this point, the spike transforms, unfolding and refolding itself, using coiled spring-like parts that start out buried at the core of the spike. The reconfigured spike hooks and docks the virus particle to the host cell. This forms a channel allowing the viral genetic material into the unsuspecting cell, in the case of COVID-19, type II lung cells. From this point onwards, most of the damage caused by COVID-19 results from the immune system going into overdrive to stop the virus from spreading.⁽⁴⁾The influx of immune cells to the infected tissue causes severe damage in the process of cleaning out the virus, infected cells, and bacterial infections with potentially lethal consequences.

Treatments

Medical therapies to treat COVI-19 evolved rapidly. Treatments include drugs that were approved by the US Food and Drug Administration (FDA) and drugs made available under FDA emergency use authorizations (EUA). The Centers for Disease Control and Prevention (CDC) has strongly encouraged clinicians, patients, and their advocates to consult the treatment guidelines published by the National Institute of Health (NIH). These guidelines are based on scientific evidence and expert opinion.⁽⁵⁾Several therapeutic modalities have been tested and deployed to treat the disease, some of which are summarized here.

Anti-virals:

Antivirals are drugs that arrest the replication of the virus. They are generally considered most effective when administered in the early phase of infection.

Remdesivir:

To date, remdesivir is currently the only antiviral approved under EUA by the FDA to treat COVID-19. The approval was based on findings that hospitalized patients who receivedremdesivir recovered faster.⁽⁶⁾Remdesivir can be administered either alone or in combination with other medications.

Molnupiravir:

An antiviral drug, previously known as EIDD-2801, appears safe and effective. Viral levels reduce to undetectable levels in COVID-19 patients after five days of administration, according to data from a US-based Phase II clinical trial. While molnupiravir is proven to inhibit coronavirus replication in infected patients, more data is required to determine whether it can prevent severe illness.⁽⁷⁾

Lopinavir/ritonavir:

Lopinavir/ritonavir are anti-human immunodeficiency virus (HIV) drugs. Both have been investigated and neither drug showed any efficacy in large randomized controlled trials in hospitalized COVID-19 patients.⁽⁸⁾

Anti-inflammatories:

One reason for mortality in COVID-19 infected patients is an overactive response by the patient’s immune system. This response leads to several inflammatory disorders, not least of which is the much publicized “cytokine storm”. The following outlines agents have been tested to dampen the inflammatory response to COVID-19.

Dexamethasone:

Dexamethasone is an anti-inflammatory corticosteroid used for many years to treat autoimmune conditions and allergic reactions. It is cheap and widely available and has been shown to reduce mortality in the sickest hospitalized patients by dampening the immune response.⁽⁹⁾ A meta-analysis study evaluating the results of seven trials shows the death rates were lower in hospitalized patients who took one of three different corticosteroids — dexamethasone, hydrocortisone, or methylprednisolone.⁽⁶⁾

Baricitinib:

Baricitinib is an anti-inflammatory drug used for the treatment of rheumatoid arthritis. In November 2020, the FDA issued an EUA to use baricitinib in combination with remdesivir in hospitalized adults and children two years and older requiring respiratory support. However, there is not enough evidence to support the use of this therapy with or without remdesivir.⁽¹⁰⁾

Antibody Based Treatments:

Antibodies are proteins generated by the immune system to help fight infections, such as viruses, by binding to and destroying them. Antibody-based treatments are likely most helpful soon after infection, rather than after the disease has progressed.

Monoclonal antibodies:

Monoclonal antibodies are synthesized in the laboratory. The FDA has approved two monoclonal antibody treatments, one single antibody from Eli Lilly, and a combination of two antibodies from Regeneron.

The Eli Lilly antibody, Bamlanivimab (LY-CoV555), works by blocking COVID-19 from entering and infecting human cells. Preliminary results indicated that patients with mild-to-moderate COVID-19 who received bamlanivimab were less likely to be hospitalized. Studies are still underway, both as a monotherapy and combination therapy. Regeneron’s treatment utilizes a combination of two monoclonal antibodies, casirivimab and imdevimab (REGN-COV2), referred to as an antibody cocktail. Preliminary trial data reported that REGN-COV2 reduced viral load and relieved symptoms sooner in non-hospitalized patients. These treatments are available for patients under EUAs, but more data is required before becoming part of routine care.⁽⁶⁾

Convalescent plasma:

One of the first groups of antibody-based treatments used convalescent plasma (plasma from recovered COVID-19 patients). This treatment involves administering plasma from a recovered individual into someone infected with the virus. Theoretically, the antibodies fromthe recovered individual neutralize the virus in the infected individual. Although the FDA issued an EUA for convalescent plasma for hospitalized patients with COVID-19, the data to date has been conflicting and inconclusive.⁽⁶⁾

Anti-coagulants:

Because of the systemic nature of COVID-19 where the circulatory system supplies all parts of the body, some COVID-19 deaths are believed to be caused by blood clots forming in major arteries and veins. A recent study has reported that full-dose blood thinners decreased the need for life support and improved outcome in hospitalized COVID-19 patients.⁽¹¹⁾ This worldwide large clinical trial, where full dose treatments were administered to moderately ill patients hospitalized for COVID-19, reduced the requirement of vital organ support—such as the need for ventilators.

In addition to some of the FDA approved drugs cited in the previous section, multiple treatments were investigated during the early phase of the COVID crisis, with varying results.⁽¹²⁾ In contrast to the overall trials for COVID-19 treatments, the programs initiated for vaccine development have been incredibly successful, surpassing all expectations.

Vaccines

From the outset of the COVID-19 pandemic, vaccines ultimately offer the most appealing and robust therapeutic modality as they prevent the disease from taking hold. This has led to a global vaccine R&D effort that is unprecedented in terms of scale and delivery. The urgency to create a vaccine for COVID‑19 led to expedited schedules that compressed the standard vaccine development timeline from years to months.

At the time of writing, three vaccines have been authorized for emergency use by the FDA in the US, with more likely to come onstream as they progress through the development pipeline. A fourth vaccine, from Oxford-AstraZeneca, has also been approved for distribution within the European Union (EU). The three vaccines approved in the US are highly effective at preventing hospitalization, death, and severe disease. Vaccines work by triggering an immune response that generate highly specific antibodies against an antigen, in the case of COVID-19, the virus spike protein expressed on the surface of the virus. Moreover, the immune system is taught to recognize the spike protein specific to the virus. If this spike protein is encountered in the future, an immune response is swiftly mounted, thus preventing escalation of the virus.

Two of the authorized vaccines, developed by both Pfizer/BioNTech and Moderna, have revolutionized a technology referred to as messenger RNA (mRNA) technology. This technology acts as a delivery system to cells within our bodies with specific instructions to carry out a specific task.⁽¹³⁾

Of importance:

mRNA vaccines do not use live virus, but rather a portion of the message encoding for the spike protein.
mRNA is produced by DNA, but does not enter the nucleus of the cell containing the DNA.
Once the mRNA vaccine finishes producing the protein that is expressed on the cell surface, it is broken down and removed by normal cellular processes.

The Johnson and Johnson (J&J) and Oxford-Astra Zeneca vaccines utilize a more conventional approach, referred to as a viral vector. This vaccine utilizes a harmless modified version of a common cold virus to deliver the gene encoding for the spike protein into the cell.⁽¹³⁾

Vaccine comparison:

Both the Pfizer/BioNTech and Moderna vaccines have been reported to confer over 94% protection rates against symptomatic COVID-19 infection.^(14,15) The single shot J&J vaccine has shown to be 85% effective at protecting against severe disease, 66% against preventing moderate to severe disease, and also appears to be effective against the South African variant of the virus (B.1.351).⁽¹⁶⁾ Although superficially the single shot J&J appears less effective, it is difficult to compare all three vaccines directly because of differences in trial design and outcomes. From a logistical point of view, the J&J vaccine is advantageous as it is a single-dose regimen that can be stored for up to three months in a refrigerator. The most recent data from the AstraZeneca phase three trial reports that the vaccine is 76% effective against symptomatic cases of the virus.⁽¹⁷⁾

Several other trials are ongoing

Several other trials are ongoing. The most important point from the information collected from 7 large efficacy trials is that all vaccines confer 100% protection against severe disease, hospitalizations, and death. Moreover, it is not just the vaccinated individual who benefits from vaccination. Most vaccines also reduce transmission of infection among people, and in so doing, help protect those who fail to mount an effective immune response to vaccines or who cannot receive the vaccinate because of their age or compromised immune system.⁽¹⁸⁾

Vaccines and viral variants:

Several variants of the virus have been reported, with the two properties causing the most concern being enhanced contagion and immune response evasion. The current vaccines were developed based on the spike protein before it contained the mutations identified in the variants. A recently published study investigated the effectiveness of the FDA approved Pfizer/BioNTech vaccine against the newly-emerged variants from the United Kingdom (UK) and South Africa (SA).⁽¹⁹⁾ While both variants are deemed more transmissible, the levels of antibody generated in response to the vaccine are so high that it seems unlikely that it will impact the overall efficacy of the vaccine for these strains. This preliminary study also highlights the ongoing evolution of COVID-19, necessitating continuous monitoring of the significances of viral mutations for vaccine efficacy. While research suggests that COVID-19 vaccines have lower efficacy against the variants, and further research is needed, the vaccines appear to provide protection against severe COVID-19.⁽²⁰⁾

Vaccine manufacturing and distribution:

The development of the vaccines, from basic R&D through human clinical trials, has been carried out within a very rapid time frame. Ramping up production, however, has been slow and cumbersome. After a slow start, Pfizer/BioNTech andModerna have raised output by gaining experience, scaling up production lines, and taking other steps like making certain raw materials on their own.⁽²¹⁾

Of the three candidates, AstraZeneca has already struck a deal with COVAX, the global initiative to distribute COVID-19 vaccines equitably. Moderna has partnered with Lonza and Catalent Inc. to manufacture and distribute millions of doses.⁽²²⁾ Moreover, a recent agreement between J&J and Catalent has secured a US FDA emergency clearance that allows Catalent’s facility to manufacture and distribute, thus aiding the vaccination supply.

Vaccination and reinfection:

The first large scale study investigating whether reinfection can occur recently reported that the vast majority of people who had COVID-19 are protected from catching it again, for at least six months.⁽²³⁾ This Danish study found that protection against repeat COVID-19 infection is both robust and detectable in the majority of individuals, 80% or more of the naturally infected population younger than 65 years. However, individuals aged 65 years and older had less than 50% protection against repeat infection, since the older age group is more susceptible serious illness. Their finding highlights the need for continued vigilance to keep themselves and others safe. This also indicates that vaccination of previously infected individuals should be done because natural protection cannot be relied upon, consistent with the general consensus that vaccines confer a level of immune response that is higher than previous COVID-19 infection. Follow-up studies will give a better idea of the duration of vaccine protection.

Conclusion

As the COVID-19 pandemic has demonstrated, it is extremely difficult to eliminate a virus from the human population once it has entered. The pandemic spread has been compounded because the virus spreads asymptomatically. That said, the virus is manageable, similar to the manner in which vaccines have worked against other preventable communicable diseases. Monitoring the protective effects of the different vaccines will likely last for several years. For now, the outlook is positive as global cases decline, the vaccines roll out, and the momentum to investigate and repurpose drugs continues.

Acknowledgments

The author is grateful to Tara Finn for the careful reading of this manuscript.

Conflict of interest

There is no conflict of interest.

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Distinctive Molecular typing of 16S rRNA of Bacillus species isolated from farm settlement.

Sun, 06 Jun 2021 00:00:00 +0000

Introduction: There are numerous methods of isolating and detecting organisms that are similar and closely related; one of the most reliable method is molecular typing of 16S rRNA. Apart from being omnipresent as a multigene family, or operons; it is evolutionarily stable; the 16S rRNA gene (1,500 bp) is large enough for informatics purposes.

Materials and Method: This study employed molecular sequencing of 16S rRNA by Sanger method to reveal the specific organisms’ nucleotides and blasting (BLASTn) to show the similarities between the resulting organisms and existing organisms. The 16S rRNA remains the best choice of identification process for bacteria because of its distinguishing sizes and evolutionary stability.

Results: All isolates were Gram positive rods and were positive in Biochemical tests such as oxidase, catalase, citrate, and protease but were in turn negative in coagulase and indole test tests. On sensitivity test; 80% of all the isolates were resistant to common antibiotics except ciprofloxacin and ceftriaxone. Based on the sequence difference in the variable region (V1) of 16S rRNA as observed from the molecular sequencing results; four isolates out of ten were identified. Six were different strains of B cereus. Others isolates include: wiedmannii, thuringensis, toyonensis and pseudomycoides. Sequence analysis of the primer annealing sites showed that there is no clear‐cut difference in the conserved region of 16S rRNA, and in the gyrB gene, between B. cereus and B. thuringiensis strains. Phylogenetic analysis showed that four isolates showed high similarity to each other; hence the limited number of deletions when subjected to alignments by maximum neighborhood joining parsimony using MEGA X software. B. toyonensis, B. wiedmannii and thuringensis were distantly related.

Introduction

Authors Pathogens cause illness and death in some countries and it also causes infections and gastrointestinal diseases in other countries thereby causing public health concern. Pathogens are organisms capable of causing diseases. Reliable methods are needed for the detection of pathogens due to pathogen evolution as a result of new human habits or new industrial practices.

Microbial classification of organisms ranges from genus to specie level depending upon the technique used either phenotypic or genotypic. Presently, molecular methods now obtain advances to allow utilization in microbiology [1]. There are numerous molecular methods which are of fast and simple application to the detection of pathogen. Among the pathogens involved in human health, Bacillus cereus is interesting due to their ability to survive in various habitats [2].

The genus Bacillus is aerobic or facultative anaerobic bacteria, gram positive spore forming rod shaped bacteria. Which can be characterized by two morphological forms, the vegetative cell which range from 1.02 to 1.2 um in width and from 3.0 to 5.0 in length, it can be straight or slightly curve, motile or non-motile, and the endospore (the non-swelling sporangium). The genus Bacillus is been characterized by the presence of endospore, which is not more than one per cell and they are resistant to many adverse environmental conditions such as heat, radiation, cold and disinfectants. It can also respire either in the presence or absence of oxygen [3]. Cell diameter of Bacillus cereus, sporangium and catalase test do not allow differentiation, where as important in differentiation among B. anthracis, B. cereus, B. thuringiensis can be considered by parasporal crystals and the presence of capsule. [4] Showed a B. thuringiensis strain capable of producing a capsule resembling that of B. anthracis. Most species of the genus display a great kind in physiological characteristics such as degradation of cellulose, starch, pectin, agar, hydrocarbons, production of enzymes and antibiotics and other characteristic such as acidophile, alkalinophile, psychrophile, and thermophile's which allows them to adapt to various environmental conditions [5]. In differentiating between species of the genus Bacillus it was difficult at early attempts when endospore formation and aerobic respiration were the main character used for classification. As reported by many authors that at molecular method level, the differentiation between B. thuringiensis and B. cereus is also very difficult.

cereus can survive at the temperature between 4°c and 55°c. The mesophile strains can grow between the temperature of 10°c and 42°c, while psychotropic strains can survive at 4°c, whereas other strains are able to grow at 52 to 55°c. B. cereus vegetative cells grow at pH between 1.0 and 5.2. Heat resistant strain can survive and multiply in wet low acid foods in temperature ranging from 5 to 52°c. The survivability of B. cereus spores at 95°c decreases when the pH level decreases from 6.2 to 4.7 [6]. B. cereus can grow in the presence of salt with concentration up to 7.5% depending on the pH value.
thuringiensis possesses a protein crystal that is toxic to insects. This toxin protein was first known as parasporal crystalline inclusion but was later referred to as π - endotoxin or in other ways known as insecticidal crystal protein [7]. Strains of B. thuringiensis bacteria possess a wide range of specificity in various orders of insects such as Lepidoptera, dipteral, coleoptera. These strains of bacteria produce crystalline proteins known as cry protein during sporulation. When B. thuringiensis infects an insects, it will cause the insect to loose appetite, enhances slow movement and over time the insect will die due to crystals of proteins that have been dissolved in the insect's stomach.

In the cultivation of vegetable crops, the plant can be attack by many types of pests. Hence, in overcoming pest attacks farmers often use pesticides that contain active synthetic materials. Many negative effects arise from the folly use of chemical pesticides. Among the negative effect is the increase of pest population, resistance, death of natural enemy population and increase in residue level on Agricultural product which makes it unsafe for public consumption [8]. Therefore, it is necessary to find an alternative method in the control of crop pest. The best alternative that can be done is to replace the chemical insecticide with biological control which involves the use of living things in the form of microorganisms. In these profiling microbial communities, the main objective is to identify which bacteria and how much they are present in the environments. Most microbial profiling methods focus on the identification and quantification of bacteria with already sequenced genomes. Further, most methods utilize information obtained from entire genomes. Homology-based methods such as [1–4] classify sequences by detecting homology in reads belonging to either an entire genome or only a small set of marker genes. Composition-based methods generally use conserved compositional features of genomes for classification and as such they utilize less computational resources.Using the 16S rRNA gene instead of whole genome information is not only computational efficient but also economical; Illumina indicated that targeted sequencing of a focused region of interest reduces sequencing costs and enables deep sequencing, compared to whole-genome sequencing. On the other hand, as observed by [8], by focusing exclusively on one gene, one might lose essential information for advanced analyses. We, however, will provide an analysis that demonstrates that at least in the context of oral microbial communities, the 16S rRNA gene retains sufficient information to allow us detect unknown bacteria

[9, 10]. This study aimed at employing 16S rRNA as an instrument of identification of seemingly close Bacillus species.

Abbreviations

BLAST, Basic Local Alignment sequence Tools; PCR, Polymerase Chains reactions; rRNA, ribosomal RNA;

Material and methods

T Sample collection. Soil samples were collected from three sources from Rice, Sugar Cane, vegetables and abandoned farmland in January 2019. The samples were labeled serially from Sample 1 to Sample 10 (S₁ to S₁₀).

Bacterial culture: A serial dilution of 10 folds was performed. Bacterial suspension was diluted (10-¹⁰) with saline water and 100 μl of bacterial suspension werespread on Nutrient Agar plate and incubated for 24 hours. Bacterial colonies were isolated and grown in Nutrient Broth and nutrient agar. Other microbiological solid agar used include: Chocolate, Blood Agar, EMB, MacConkey, Simon citrate, MRS Agar. Bacteria were characterized by conventional technique by the use of morphological appearance and performance on biochemical analysis [11].

Identification of bacteria:The identification of bacteria was based on morphological characteristics and biochemical tests carried out on the isolates. Morphological characteristics observed for each bacteria colony after 24 h of growth included colony appearance; cell shape, color, optical characteristics, consistency, colonial appearance and pigmentation. Biochemical characterizations were performed according to the method of [12]

Catalase test: A small quantity of 24 h old culture was transferred into a drop of 3% Hydrogen peroxide solution on a clean slide with the aid of sterile inoculating loop. Gas seen as white froth indicates the presence of catalase enzyme [13] on the isolates.

DNA Extraction Processes

The extraction processes was in four phase which are:

Collection of cell, lyses of cell, Collection of DNA by phenol, Concentration and purification of DNA.

Collection of cell: the pure colonyof the bacteria culture was inoculated into a prepared sterile nutrient broth. After growth is confirmed by the turbidity of the culture, 1.5ml of the culture was taken into a centrifuge tube and was centrifuge at 5000 rpm for 5 minutes; the supernatant layer was discarded leaving the sediment.

Lyses of cell: 400 microns of lyses buffer is added to the sediment and was mixed thoroughly and allow to stand for five minutes at room temperature (25°c). 200 microns of Sodium Dodecyl Sulfate (SDS) solution was added for protein lyses and was mixed gently and incubated at 65°c for 10 minutes.

Collection of DNA by phenol; 500 microns of phenol chloroform was added to the solution for the separation of DNA, it was mixed completely and centrifuge at 10,000 rpm for 10 minutes. The white pallet seen at the top of the tube after centrifugation is separated into another sterile tube and 1micron of Isopropanol is added and incubated for 1hour at -20°c for precipitation of DNA. The DNA is seen as a colorless liquid in the solution.

Concentration and purification of DNA: the solution was centrifuge at 10,000 rpm for 10 minutes. The supernatant layer was discarded and the remaining DNA pellets was washed with 1micron of 17% ethanol, mixed and centrifuge at 10,000 rpm for 10 minutes. The supernatant layer was discarded and air dried. 60 micron TE. Buffer was added for further dissolving of the DNA which was later stored at -40°c until it was required for use [14].

PCR Amplification

This requires the use of primers (Forward and Reverse), polymerase enzyme, a template DNA and the d pieces which includedddATP, ddGTP and ddTTP, ddNTP. All this are called the master mix.

The PCR reactions consist of three main cycles.

The DNA sample was heated at 94⁰c to separate the two template of the DNA strand which was bonded by a hydrogen bond. Once both strand are separated the temperature is reduced to 57⁰c (Annealing temperature). This temperature allows the binding of the forward and reverse primers to the template DNA. After binding the temperature is raised back to 72⁰c which leads to the activation of polymerase enzyme and its start adding d NTPs to the DNA leading to the synthesize of new strands. The cycles were repeated several times in order to obtain millions of the copies of the target DNA [15].

Preparation of Agarose Gel

One gram (1 g) of agarose for DNA was measured or 2 g of agarose powdered will be measured for PCR analysis. This done by mixing the agarose powder with 100 ml 1×TAE in a microwaveable flask and microwaved for 1-3 minutes until the agarose is completely dissolved (do not over boil the solution as some of the buffer will evaporate) and thus alter the final percentage of the agarose in the gel. Allow the agarose solution to cool down to about 50°c then after five minutes 10µL was added to EZ vision DNA stain. EZ vision binds to the DNA and allows one to easily visualize the DNA under ultra violet (UV) light. The agarose was poured into the gel tray with the well comb firmly in place and this was placed in newly poured gel at 4°c for 10-15 mins or it sit at room temperature for 20-30 mins, until it has completely solidified[16].

Loading and Running of samples on Agarose gel

The agarose gel was placed into the chamber, and the process of electrophoresis commenced with running buffer introduced into the reservoir at the end of the chamber until it the buffer covered at least 2millimeter of the gel. It is advisable to place samples to be loaded in the correct order according to the lanes they are assigned to be running. When loading the samples keep the pipette tip perpendicular to the row of the wells as by supporting your accustomed hand with the second hand; this will reduce the risk of accidentally puncturing the wells with the tip. Lower the tip of the pipette until it breaks the surface of the buffer and is located just above the well. Once all the samples have been loaded it is advised to always avoid any movement of the gel chamber. This might result in the sample spilling into adjacent well. Place the lid on the gel chamber with the terminal correctly positioned to the matching electrodes on the gel chamber black to black and red to red. Remember that DNA is negatively charged hence the movement of the electric current from negatively charged to the positively charged depending on the bandwidth in Kilobytes. Once the electrode is connected to the power supply, switch ON the power supply then set the correct constant voltage (100) and stopwatch for proper time. Press the start button to begin the flow of current that will separate the DNA fragment.After few minutes the samples begins to migrate from the wells into the gel. As the DNA runs, the diaphragm moves from the negative electrode towards the positive electrode [17].

PCR mix Components and Sanger Sequencing

This is made up of primers which is both Forward and Reverse, the polymerase enzyme (Taq), a template DNA and the pieces of nucleotides which include: ddNTP, ddATP, ddGTP and ddTTP. Note that the specific Primer’s sequences for bacterial identification is: 785F 5' (GGA TTA GAT ACC CTG GTA) 3', 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3', 907R 5' (CCG TCA ATT CMT TTR AGT TT) 3', 1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3' in Sanger Sequencing techniques.

BLAST

The resulting genomic sequence were assembled and submitted in GenBank at NCBI for assignment of accession numbers. The resultant assertion numbers were subjected to homology search by using Basic Local Alignment Search Tool (BLAST) as NCBI with the assertion number MW362290, MW362291, MW362292, MW362293, MW362294 and MW362295 respectively. Whereas, the other isolates’ accession numbers were retrieved from NCBI GenBank which are:AB 738796.1, JH792136.1, MW 015768.1 and MG745385.1.MEGA 5.2 software was used for the construction of phylogenetic tree and phylogenetic analysis.

All the organisms possess 100% identities, 0% gaps and 0.0% E.value which indicated that the organisms are closely related to the existing organisms. The use of 16S rRNA is the best identification process for bacteria because 16S rRNA gene has a distinguishing size of about 500 bases until 1500bp. Rather than using 23S rRNA which is of higher variation, The 16S rRNA is adopted in prokaryotes. 18S rRNA is used for identification in Eukaryotes

Results

The results of both the conventional morphological and cultural identification was correlated with the molecular sequencing results. Six isolates were confirmed B. cereus species while the other four isolates were. B. wiedmannii, B. thuringiensis, B. toyonensis and B. pseudomycoides.The 16S rRNA sequence of six isolates MW 362290.1- MW362295.1 were assigned accession numbers and deposited in the GenBank while the other four sequences were aligned to those available in the NCBI database. The alignment results showed closely relatedness to LT844650.1with an identity of 100% to 92.2% as above. The six isolates of Bacillus cereus great evolutionary relatedness as shown in the phylogenetic tree constructed using MEGA X software.

Results

Discussion

The results obtained in this study is consistent with the previous studies in other countries22,23 The results of the phylogenetic analysis of the 16S rRNA isolate of in this study was similar to the housekeeping genes proposed by [18, 19]. In comparing this study with the earlier study, B. cereus group comprising other species of Bacillus was hypothesized to be considered to form a single species with different ecotypes and pathotype. This study was able to phenotypically differentiated B. thuringiensis, B. pseudomycoides, B. toyonensis, B. wiedmannii and B. cereus sensu strito. Despite differences at the colonial appearance level, the 16S rRNA sequences have homology ranging from 100% to 92% providing insufficient resolution at the species level [6, 7, 18].After analysis through various methods, the strain was identified as Gram-positive bacteria of Bacillus cereus with a homology of 99.4%. Cohan [20] demonstrated that 95–99% of the similarity of 16S rRNA gene sequence between two bacteria hints towards a similar species while >99% indicates the same bacteria.The phylogenetic tree showed that B. toyonensis, B. thuringiensis and B. wiedmanniiare the outgroups of B. cereus

group while B. pseudomycoides are most closely related to B. cereus group [19, 21, 22].

Conclusion

In the area of molecular epidemiology, genotypic typing method has greatly increased our ability to differentiate between micro-organisms at the intra and interspecies levels and have become an essential and powerful tool. Phenotypic method will still remain important in diagnostic microbiology and genotypic method will become increasingly popular.

After analysis through various methods, the strain was identified as Gram-positive bacteria of Bacillus cereus with a homology of between 100% and 92.3%.

Acknowledgments

Collate acknowledgments in a separate section at the end of the article before the references, not as a footnote to the title. Use the unnumbered Acknowledgements Head style for the Acknowledgments heading. List here those individuals who provided help during the research.

Conflicts of interest

The Authors declare that there is no conflict of interest.

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Prediction of Multi-Epitopic Domains of a Putative Oral Vaccine against Hepatitis C Virus

Wed, 21 Jul 2021 00:00:00 +0000

For vaccine development, triggering an immune response is desired. Designing and assessing vaccine candidates for the appropriate immune response is critical for their success. Hepatitis C virus is the major cause of liver disease. Anti HCV vaccines if designed is rational decision to reinforce specific T-cell as a crucial aspect of effective antiviral treatment. This study explored the use of bioinformatics tools by retrieval of twenty (20) HCV proteins which were selected for vaccine design. These were retrieved from UniProt server based on their antigenicity, virulence, subcellular localization, essentiality non-homology and other physical parameters, including, TM helices, and relative molecular mass. BLASTp revealed 80% non-identity with Homo sapiens genes. The Epitopes obtained include: Q3S781_9HEPC52-71, POLG_HCVBK442-461, POLG_HCVJA2-21, POLG_HCVJ177-95, POLG_HCVCO445-464, POLG_HCVR61107-1126, POLG_HCVJP47-66, POLG_HCVTW664-683, POLG_HCVTR446-465, LTOR5_HUMAN23-42, POLG_HCVT5100-119, POLG_HCVJT77-96, HOIL1_HUMAN169-188, POLG_HCVJ4644-663, POLG_HCVJ847-66, TFB2M_HUMAN49-68, RSF1_HUMAN138-157, A8DGK3_9HEPC77-96, A8DHN1_9HEPC54-73, and A8DFL0_9HEPC2-21. An antigenicity score of 0.6004 was obtained with the use of VaxiJen server. The allergenicity prediction showed that the vaccine is not allergenic with the use of AllerTOP v.2.0 and AlgPred servers. The molecular weights and theoretical pI of protein were 45.1 kDa and 10.24 kDa respectively. A potentially suitable vaccine candidate with multivariant regions and immunogenic which could be antagonistic to HCV was designed.