Lipid nanoparticle library towards development next generation genomic medicines

Ionizable amino lipids are a major constituent of the lipid nanoparticles for delivering nucleic acid therapeutics (e.g., DLin-MC3-DMA in ONPATTRO , ALC-0315 in Comirnaty , SM-102 in Spikevax ). Scarcity of lipids that are suitable for cell therapy, vaccination, and gene therapies continue to be a problem in advancing many potential diagnostic/therapeutic/vaccine candidates to the clinic. Herein, we describe the development of novel ionizable lipids to be used as functional excipients for designing vehicles for nucleic acid therapeutics/vaccines in vivo or ex vivo use in cell therapy applications. We first studied the transfection efficiency (TE) of LNP-based mRNA formulations of these ionizable lipid candidates in primary human T cells and established a workflow for engineering of primary immune T cells. We then adapted this workflow towards bioengineering of CAR constructs to T cells towards non-viral CAR T therapy. Lipids were also tested in rodents for vaccine applications using self-amplifying RNA (saRNA) encoding various antigens. We have then evaluated various ionizable lipid candidates and their biodistribution along with the mRNA/DNA translation exploration using various LNP compositions. Further, using ionizable lipids from the library, we have shown gene editing of various targets in rodents. We believe that these studies will pave the path to the advancement in nucleic acid based therapeutics and vaccines, or cell gene therapy agents for early diagnosis and detection of cancer, and for targeted genomic medicines towards cancer treatment and diagnosis.

Monkeypox (mpox) is a new problem on a global scale for humanity.

Purpose - to draw attention to an infection that was little known, but has now become a global problem for society;to familiarize readers with the peculiarities of the 2022 monkeypox outbreak and to increase the level of alertness of doctors to this disease. Monkeypox is a global problem because the disease is spreading rapidly, covering 111 countries. Three cases were diagnosed in Ukraine. It is predominantly a self-limited infection, but there are severe and deadly complications. The lethality of this disease ranges from 0% to 11%. The course of the disease is more severe in children and people with reduced immunity. Vertical transmission of the virus from mother to child is possible, resulting in congenital monkeypox. Monkeypox is a zoonotic disease and its natural reservoir is not exactly known, but rodents are most likely to act. In most cases, person-to-person transmission of the virus occurs through close skin to skin contact, often during sexual intercourse. At the beginning of the outbreak 98% of cases of disease were was diagnosed in homo- and bisexuals. Airborne transmission is also possible. Infection is possible through close contact with infectious skin lesions. Clinically, the initial period resembles influenza, but lymphadenopathy is characteristic, which is considered a pathognomonic symptom of mpox. The rash is similar to that of chickenpox, but with more prevalent location on palms and soles than in chickenpox. In the presence of a vesicular rash in a patient, it is necessary to exclude monkeypox. PCR diagnostics of the virus in samples of vesicles or crusts has the greatest diagnostic value. Hygienic skin care is important. Antiviral drugs (tecovirimat, brincidofovir) are recommended only in severe cases. To reduce the spread of infection, international rules apply as for other infections, such as COVID-19. The monkeypox virus vaccine is recommended primarily for groups at risk of infection, including medical personnel who may come into contact with the patient or samples for laboratory testing. Being aware for this infection, following international health regulations, it is possible to prevent the further spread of monkeypox.Copyright © 2023 Tomsk State University. All rights reserved.

COVID-associated pernio: Mechanistic insights to an abortive, seronegative SARS-CoV-2 infection

Introduction: SARS-CoV-2 replicates primarily in the airways but generates a systemic immune response mediated by Type I interferons (IFN-I). Pernio is a rare skin manifestation of disorders characterized by excessive IFN-I signalling. Although pernio increased in incidence during the pandemic, the relationship to SARS-CoV-2 remains controversial. Because of the pivotal nature of interferons in COVID-19 outcomes, pernio offers a window to investigate the biology underlying host resiliency to SARS-CoV-2 infection. Method(s): To further assess COVID-associated pernio, we characterized clinical samples from affected patients across 4 waves of the pandemic and investigated mechanistic feasibility in a rodent model. Patients were followed longitudinally with banking of blood and tissue. Golden hamsters were mock-treated or intra-nasally infected with SARS-CoV-2 and harvested at 3-and 30-days post-infection. Result(s): In affected tissue, immunophenotyping utilizing multiplex immunohistochemistry profiled a robust IFN-1 signature characterized by plasmacytoid dendritic cell activation. Viral RNA was detectable in a subset of cases using in situ hybridization for the SARS-CoV-2 S gene transcript. Profiling of the systemic immune response did not reveal a durable type 1 interferon signature. Consistent with previous literature, antibody and T-cell specific responses to SARS-CoV-2 were not detected. Nasopharyngeal SARS-CoV-2 inoculation in hamsters resulted in rapid dissemination of viral RNA and the generation of an IFN-I response that were both detectable in the paws of infected animals. Conclusion(s): Our data support a durable local IFN signature, with direct evidence of viral SARS-CoV-2 RNA in acral skin and suggest that COVID-associated pernio results from an abortive, seronegative SARS-CoV-2 infection.

Covid-19: A Multi-Host Pandemic

SARS-CoV-2, the causal agent of the COVID-19 pandemic, is related to a group of viruses (Sarbecovirus) that circulate in horseshoe bats. Its origin is still uncertain, as there is lack of an identifiable intermediate host species for the proximal animal ancestor of SARS-CoV-2. Irrespective of its origin, SARS-CoV-2 has been shown to replicate in many mammalian species. So far, over forty species have been found to be susceptible to SARS-CoV-2 infection, and natural infections have been documented in at least 23 species of distant mammalian orders, including Primata, Rodentia, Carnivora, and Arthiodactyla. In two of those species, minks and white tailed deer, continued transmission among conspecifics occurred following introduction of SARS-CoV-2 from humans, at a rate which makes mink farms and deer populations suitable compartments where the virus may be maintained and evolve, and then perhaps spill back to humans or other animals as a new variant, as suggested by molecular evidence. Considering the above, what is truly unique about this pandemic, and adds a major obstacle to attain its control, is its multi-host nature. This is another compelling example of the relevance of the 'One Health' approach. This approach recognizes the inextricable links between people and nature, and visualizes the health and disease phenomenon from an integrative perspective. The COVID-19 pandemic urges us to acknowledge the interconnection between people and the remaining forms of life, and with the environments they share, and demonstrates that the improvement of global health needs a collaborative, multisectoral, and transdisciplinary approach, acting at the local, regional and global levels. This concept becomes paramount when taking into account that most diseases affecting humans in the last decades -not only COVID-19 - have been caused by pathogens originated in animals.Copyright © 2023

Human coronaviruses in the 'One Health' context

Seven human coronaviruses have been identified so far: four seasonal coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1) and three novel coronaviruses (SARS-CoV, MERS-CoV, SARS-CoV-2). While seasonal coronaviruses cause only mild symptoms, novel coronaviruses cause severe and potentially fatal infections. All known coronaviruses originated in animals. Bats are considered as an origin for the majority of coronaviruses capable of infecting humans;however, rodents are proposed as natural hosts for HCoV-OC43 and HCoV-HKU1. Different animal species could serve as intermediate hosts including alpacas (HCoV-229E), livestock (HCoV-OC43), civet cats (SARS-CoV), camels (MERS-CoV), and pangolins (SARS-CoV-2). In Croatia, SARS-CoV-2 was detected in humans, pet animals, wildlife, and the environment. The COVID-19 pandemic has highlighted the role of the 'One Health' approach in the surveillance of zoonotic diseases.Copyright © 2022, University Hospital of Infectious Diseases. All rights reserved.

Insight of Zoonotic Viruses at Human-Animal Interfaces in Cambodia

*Presenting author Emerging infectious diseases have been causing outbreaks in humans for centuries and most infectious diseases originate in animals. Re-emerging zoonotic pathogens are rapidly increasing in prevalence or geographic range and causing a significant and growing threat to global health. The present work provides an insight of zoonotic viruses risk at human-bat/rodent interfaces in Cambodia. We conducted studies to investigate the circulation of zoonotic viruses and the risk of exposure in human living at the interfaces with bats and rodents. Rodent's samples were collected in rural and urban areas of Cambodia. Organs were tested for Hantavirus, Orthohepevirus species C and Arenavirus. Bat's samples were collected in Steung Treng for Sarbecovirus and in Battambang and Kandal for Nipah virus detection. People working/living at the human-animal interfaces were screened for IgG antibodies. In rodents (750), hantavirus was detected in 3.3% rodents from urban areas only. Seoul orthohantavirus was the most predominant virus followed by Thottapalayam virus. HEV-C was detected only in rodents from urban settings (1.8%). Arenavirus was detected in both rural (6.8%) and urban (2.5%) areas. In humans (788), the seroprevalence of IgG antibodies against hantavirus, HEV-A and Arenavirus was 10.0%, 24% and 23.4% respectively. NiV was detected in flying fox's urines collected between 2013-2016 in Kandal (0.63%) and in Battambang (1.03%). Blood samples collected in both provinces were negative for NiV antibodies. SARS-CoV-2 related virus was detected in Rhinolphus shameli in Steung Treng in 2010, 2020 and 2021. Blood samples from people living at the vicinity of positive bats were positive for antibodies against CoV (7.7%), but no specific neutralizing SARS-CoV2 antibodies were detected. Our studies provided insight of the risk of zoonoses in Cambodia and highlighted the importance of zoonotic surveillance and further One Health effort to prevent, detect, and respond to future cross-species transmission.Copyright © 2023

Zooanthropogenic potential of SARS-CoV-2 virus: Implications for vaccine-mediated immunity

The human pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that started in December, 2019 is still continuing in various parts of the world. The SARS-CoV-2 has evolved through sporadic mutations and recombination events and the emergence of alternate variants following adaptations in humans and human-to-animal transmission (zooanthraponosis) has raised concerns over the efficacy of vaccines against new variants. The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV- 2-related viruses in bats and pangolins. A recent report of back-andforth transmission of SARS-CoV-2 between humans and minks on mink farms in the Netherlands has sparked widespread interest in zooanthroponotic transmission of SARS-CoV-2 followed by reemergence to infect human populations. The risk of animal to human transmission depends on virus-host interaction in susceptible species that may be short-term or long term risks. The short term risk might be due to infection to humans during the viremic stage in susceptible animals. The long term risk might be either due to persistence of the virus at population level or latency of infection leading to risk of evolution and re-emergence of the virus. Experimental studies have identified a range of animals that are susceptible and permissive to SARS-CoV-2 infection viz. cats, ferrets, hamsters, mink, non-human primates, tree shrews, raccoon dogs, fruit bats, and rabbits. The health impacts of SARS-CoV-2 infection in animals are unknown and it is likely that other susceptible species have not been discovered yet. Apart from farmed animals, stray cats and rodents have been identified as a potential opportunity for ongoing transmission in intense farming situations. Recognizing animal species that are most susceptible to infection is the first step in preventing ongoing transmission from humans. Minimizing the risk of zooanthraponosis requires multi-sectoral coordination that includes implementation of strict biosecurity measures such as controlled access to farms that house susceptible animals, bio-secure entry and exit protocols, disinfection protocols in farm, down time for animal transport vehicles and daily assessments of human handlers for exposure to SARS-CoV- 2. Hence, active surveillance in animal species that are prioritized based on risk assessment need to be initiated in coordination with health and environment sectors for early identification of emerging and re-emerging variants of SARS-CoV-2 virus in animals.

A Review on Recent Monkey Pox Outbreak in 2022

Monkey pox, a zoonotic disease with clinical symptoms resembling smallpox, unexpectedly broke out and spread over the world after the outbreak of COVID-19, severely affecting several of the continents of the world. Monkey pox is currently a member of the genus otrhopox virus, which is a member of the sub family chorodoxvirinae. According to the available knowledge, small mammals and rodents have all been identified as potential sources of the monkey [ox virus]. The disease is characterized by a short febrile illness with lymphadenopathy followed by a rash which spreads centrifugally and passes through phases of macules, papules, vesicles, and pustules. Recovery occurs in most patients within 2-4 wk. Complications are more likely in children, pregnant women, and the immunocompromised. Specific diagnosis is by detection of viral DNA by PCR.Tecovirimat, brincidofovir, and cidofoviir are the medications used to treat monkey pox, immunoglobulin and new compounds are the vaccinations. This review will introduce a general overview of MPXV and describe the epidemiology, clinical features, evaluation, and treatment of monkey pox patients.Copyright © Journal of Global Trends in Pharmaceutical Sciences.

Using ex vivo bioengineered lungs to model pathologies and screening therapeutics

Introduction: More than 8 million lives are claimed annually by various respiratory diseases including lung cancer. While therapeutics is the first line of defence, treatment failure always remains challenging and research studies face a lag of transition from preclinical to clinical phase. This is partly due to the inadequate representation of the preclinical models in clinical trials. In this proof-of-concept study, we sought to use an ex-vivo model to identify lung pathologies and therapeutically screen them in a rodent model. Method(s): Briefly, the heart-lung tissues were extracted and decellularized using a detergent-based decellularization technique. Subsequently, lungs were seeded and cultured (6-10 days) with human cell lines: BEAS-2B, A549, and Calu3, demonstrating healthy lung, cancerous state, and congenital pathologies (cystic fibrosis), respectively. By altering the cultural conditions and exploiting the unique characteristics of these cell lines, we were able to model a variety of novel pathological models in ex vivo, such as advanced-stage solid tumours and the primary phase of infection via SARS-COV2. We also validated the above-mentioned observations by histology and immunofluorescence staining. Another novel part of our study includes a qualitative screening of efficacy and impact of important Therapeutics (anti-neoplastic)- Cisplatin and Wogonin, in our cancer models. Result(s): Using A549 and BEAS-2B cells, we were able to model different stages of Non-small cell lung cancer, qualitatively validated the resemblance to clinical samples and monitor the impact of different therapeutics on these models. The qualitative assessment also demonstrated different levels of cell death depending on the efficacy of the drugs. Contribution to research : Collectively this study demonstrates the remarkable versatility and strength of the ex vivo model in representing important lung pathologies and screening therapeutics in the preclinical phase.

Novel Size-Variable Dedicated Rodent Oxygenator for ECLS Animal Models-Introduction of the "RatOx" Oxygenator and Preliminary In Vitro Results.

The overall survival rate of extracorporeal life support (ECLS) remains at 60%. Research and development has been slow, in part due to the lack of sophisticated experimental models. This publication introduces a dedicated rodent oxygenator ("RatOx") and presents preliminary in vitro classification tests. The RatOx has an adaptable fiber module size for various rodent models. Gas transfer performances over the fiber module for different blood flows and fiber module sizes were tested according to DIN EN ISO 7199. At the maximum possible amount of effective fiber surface area and a blood flow of 100 mL/min, the oxygenator performance was tested to a maximum of 6.27 mL O2/min and 8.2 mL CO2/min, respectively. The priming volume for the largest fiber module is 5.4 mL, while the smallest possible configuration with a single fiber mat layer has a priming volume of 1.1 mL. The novel RatOx ECLS system has been evaluated in vitro and has demonstrated a high degree of compliance with all pre-defined functional criteria for rodent-sized animal models. We intend for the RatOx to become a standard testing platform for scientific studies on ECLS therapy and technology.

A historical review of pandemics with a dermatological perspective

We present a literature review of dermatology features in historical pandemics. A pandemic is an epidemic occurring worldwide, or over a very wide area, crossing international boundaries and affecting a large number of people. Smallpox was the first documented pandemic, around 10 000 BC, spread by the inhalation of airborne droplets. A few days after an initial high fever, headache and fatigue, a mucocutaneous maculopapular eruption appeared, which then developed pustules and erosions. The last outbreak occurred in the USA in 1949. Smallpox was eradicated in 1980, following a vaccination programme. Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), an ongoing global pandemic. The earliest documentations were 3300 years ago. In 2020, the World Health Organization (WHO) provisionally estimated 1.5 million deaths globally. Most commonly affecting the lungs, cutaneous TB may present with inflammatory papules, plaques, suppurative nodules and chronic ulcers. Requiring long, complex antibiotic regimens, multidrug resistant TB is an increasing problem. Now extremely rare, yet still with recent outbreaks in 2021 in Madagascar, bubonic plague arrived in Europe in 1346 causing 75-200 million deaths. It is caused by the bacterium Yersinia pestis, transmitted through fleas that have fed on infected rodents. Clinical features include papules, pustules, ulcers and eschars, tender lymphadenopathy and systemic symptoms, and it responds to antibiotics. Syphilis, caused by the bacterium Treponema pallidum, is sexually transmitted. The first known outbreak was during warfare in 1494-5 in Naples, Italy. In 2020, the WHO estimated that, globally, seven million people had new infections. Primary syphilis typically produces a painless, genital ulcer (or chancre). Secondary syphilis presents with a nonitchy, maculopapular erythema over the trunk, palms and soles. Early recognition and antibiotic treatment usually lead to good outcomes. Estimated by the WHO to affect 37.7 million people in 2020, HIV is thought to have mutated from simian immunodeficiency virus by the 1960s in sub-Saharan Africa, spreading to the Caribbean and USA by the late 1960s. Initial symptoms include a fever, headache and lymphadenopathy. Dermatological features are common, including opportunistic cutaneous infections, nonspecific exanthemas, seborrhoeic dermatitis and Kaposi sarcoma. Advances in antiretroviral therapies mean people with HIV can have an excellent prognosis, although the WHO estimated in 2020 that more than 200 000 people with HIV died from concomitant TB. Since 2019, COVID-19 has had a considerable global impact on healthcare. With more than 300 million cases and 5.5 million deaths to date, some services have been overwhelmed owing to large case numbers, variable vaccine uptake, workplace changes to reduce transmission and staff shortages. Cutaneous features include perniosis, urticarial, purpuric, vesicular or maculopapular eruptions. Pandemics throughout history have been repeatedly shown to present with an element of skin involvement. We can utilize this to promote education and early recognition of these features, to facilitate diagnosis and raise awareness of the potential complications of serious diseases.

Living with rodent pests: Unifying stakeholder interests to prioritise pest management in rural Madagascar

Rodent pests can have major social, economic, and environmental impacts. Their management, therefore, represents a complex socio-ecological problem involving a network of stakeholders from across different sectors, with diverging and sometimes competing interests. Failure to incorporate stakeholder interests can result in ineffective or unsustainable management programmes, with unintended negative consequences for people and nature.Participatory approaches to decision-making have been proposed as suitable strategies to tackle complex problems, yet, these processes are often considered too difficult, costly, or time-consuming to implement.To facilitate a participatory approach to rodent control in Madagascar, we identified and mapped key stakeholders and developed a multisector framework for guiding rodent management programmes based on current literature and expert recommendations. We then carried out interviews and focus groups with stakeholders and end-users to validate the final framework.The final framework unifies stakeholder interests around the dimensions of People, Resources, Knowledge and Power. Combined application of the stakeholder map and framework provides decision-makers with the tools to identify stakeholder interests;to explore areas of conflict, as well as areas of agreement;and to ensure that these are addressed within the design of control programmes. As an assessment tool, the framework can also be used to evaluate the responsiveness of programmes to the needs of different stakeholders and assess whether objectives are being reached.We recommend the application of the stakeholder map and framework to encourage and strengthen participatory approaches aimed at rodent pest control. Due to the inclusive and interdisciplinary nature of the framework, it can be applied to address numerous complex social, environmental, and economic issues across scales, sectors, and systems.Read the free Plain Language Summary for this article on the Journal blog.

Aldosterone as a possible contributor to eye diseases.

Aldosterone, an effector molecule of the renin-angiotensin-aldosterone system (RAAS), has been receiving more attention in the field of ophthalmology because of its possible role in the pathogenesis of various eye diseases or abnormalities; potentially, it may even become a target for their treatment. Primary aldosteronism, a typical model of a systemic aldosterone excess, may cause vision loss due to various ocular diseases, such as retinal vein occlusion, central serous chorioretinopathy, and possibly, glaucoma. RAAS components are present in various parts and types of cells present in the eye. Investigations of local RAAS in various animal models of diabetic macular edema, retinal vein occlusion, retinopathy of prematurity, central serous chorioretinopathy, and glaucoma have found evidence that aldosterone or mineralocorticoid receptors may exacerbate the pathology of these disorders. Further studies are needed to elucidate whether the modulation of aldosterone or mineralocorticoid receptors is an effective treatment for preventing vision loss in patients with eye diseases.

Human-animal spillover of SARS-CoV-2: Implications for public health

COVID-19 pandemic caused by the recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV-2) is a significant public health catastrophe in a century. While the precise origin of SARS-CoV-2, and its mode of introduction into the human population, is not yet fully resolved, there is evidence that SARS-CoV-2 originated from bats. As zoonotic viruses infecting humans can spill back into susceptible animal hosts, SARS-CoV-2 has demonstrated the ability to infect many nonhuman animal host species. The list of animal species susceptible to SARS-CoV-2 infection continues to grow and includes domestic animals, primates, pet animals, and zoo animals. In addition, based on the ability of the spike protein to bind to the ACE-2 receptor, computational predictions have identified dozens of additional possible animal hosts for SARS-CoV-2. In addition, there are multiple reports of human infections from SARS-CoV-2 infected animals. We discovered widespread natural infection of wild white-tailed deer with SARS-CoV-2 in the USA, suggesting their role as a potential SARS-CoV-2 reservoir. Establishing an animal reservoir could facilitate the continued circulation of SARS-CoV-2 independent of circulation in humans. In addition, deer could pass on the infection to other susceptible wild animals such as rodents, foxes, and raccoons resulting in the establishment of SARS-CoV-2 enzootic transmission cycles. Such a scenario could result in virus adaptation and the emergence of novel variants that could escape the protection of current human SARS-CoV-2 vaccines. This presentation will discuss our recent findings on natural SARS-CoV-2 infection of deer and the long-term implications of human-animal-human spillover of SARS-CoV-2.

Chapter 7 - Pulling it all together: where do we go from here?

Coronaviruses are Baltimore Class I viruses of the family Coronaviridae. Similarities and differences to other members of these groups are discussed. Proposed reservoir/intermediate hosts of severe acute respiratory system coronavirus (SARS-CoV), Middle Eastern respiratory system coronavirus, and SARS-CoV-2 are presented. Bats appear to be reservoir hosts for these and some animal coronaviruses. Other potential reservoir/intermediate hosts of pathogenic coronaviruses are presented, with particular emphasis on rodents and birds. Potential methods to predict or prevent future pandemics include the One Health Approach and SpillOver. Factors driving epidemics and pandemics are discussed, particularly microbial, host-related, and environmental factors as well as ‘The Human Factor,’ medical and behavioral interventions that decrease disease spread and severity. The author’s vision for Infectious Disease Centers (IDCs), similar to Ebola Centers, is presented. IDCs would respond to a broad range of infectious diseases, utilizing separated, negative-pressure areas of existing hospitals with specialized, trained healthcare personnel, microbiologists, public health officials, and lab technicians on call. The proposed IDCs would have stockpiles of personal protective equipment (PPE), equipment, and laboratory facilities on hand to respond to a range of infections. Equipment could include ventilators, autoclaves, dialysis equipment, and three-dimensional printers. The latter was used to produce PPE and ventilators during the COVID-19 pandemic. Other innovative plans would be encouraged, such as the conversions of a deck of a long-distance Italian ferry for patients needing an intermediate level of care during the COVID-19 pandemic. Problems associated with infectious disease epidemics in developing countries are examined, with suggestions for the inclusion of appropriate personnel, such as local cultural experts and interpreters, as well as innovative planners and, perhaps, 3-D printers.

Chapter 5 - Coronaviruses of wild and semidomesticated animals with the potential for zoonotic transmission

Coronaviruses are present in most animal species. Some animals may then serve as a reservoir or intermediate hosts of viruses causing mild or severe to fatal diseases in humans and other animals. Infected humans may also transmit coronaviruses, such as severe acute respiratory syndrome virus (SARS-CoV)-2, to animals, including captive endangered animal species. This chapter focuses on coronaviruses of wild and semidomesticated animals, including viruses from bats, rodents, nonhuman primates, ferrets, minks, and rabbits. The ability of coronaviruses to rapidly mutate and to exchange their genetic material with other coronaviruses leads to the production of variants able to infect and adapt to new host species. Special attention is given to coronaviruses of bats and rodents since they appear to have hosted ancestral coronaviruses that indirectly lead to zoonotic transmission of highly pathogenic human viruses, including SARS-CoV, the closely related SARS-CoV-2, and Middle East respiratory syndrome virus. The RNA genomes of several bat coronaviruses, such as WIV1 and WIV16, are very similar to SARS-CoV. Coronaviruses in animals primarily cause severe disease in the respiratory, central nervous, and digestive systems but may damage other organ systems as well. Further studies on wildlife coronaviruses are advisable to avoid human epidemics or pandemics as well as to protect endangered animal species.

Mutagenicity of N-Hydroxycytidine and Monlnupiravir in E. Coli and Mammalian Cell Cultures

Molnupiravir (MOV) has received FDA's Emergency Use Authorization for the treatment of COVID-19, which is caused by SARS-CoV-2 infection. MOV is a prodrug of the ribonucleoside analog, Nhydroxycytidine (NHC). Upon phosphorylation, NHC incorporates into nascent viral RNA during replication triggering “catastrophic” mutation of the viral genome. However, NHC can also enter the deoxy-ribonucleotide pool, become incorporated into DNA, and cause DNA mutations. In nonclinical safety assessments, MOV was positive (i.e., mutagenic) in the Ames assay but negative in regulatory in vitro and in vivo micronucleus assays. Multiple in vitro studies conducted in bacteriophages, bacteria, fungi, and mammalian cells have reported that NHC can induce DNA mutations, mainly A:T>G:C transitions. We used a recently developed error-corrected wholegenome sequencing technique for detecting mutations induced by MOV and NHC in cultures of E. coli, mouse L5178YTk+/-, and human TK6 cells. Treatment of bacterial and mammalian cultures (for 4 hours and 5 days, respectively) with either MOV or NHC increased mutation frequencies in a dose-dependent manner in all three models. The majority of induced mutations were A:T>G:C, consistent with the type of mutation caused by incorporation of dNHC opposite to dA in the first round of DNA replication and incorporation of dG opposite to dNHC in the subsequent round(s) of DNA replication. Trinucleotide mutational signatures in MOV/NHC-treated cells were similar in mouse and human cells and different from the background spontaneous mutational signatures in parental cell cultures. The specific mutational signature was evident in mammalian cells exposed to NHC concentrations comparable to those observed in the plasma of human subjects who received clinical doses of MOV. This data indicates more well-controlled rodent and clinical studies of MOV/NHC-induced mutagenicity should be done in the interest of public health safety.

Angiotensin converting enzyme 2 in the gastrointestinal tract: binding with coronavirus and its consequences

Introduction: Angiotensin converting enzyme 2 (ACE2) is expressed in several cell types in the body including the gastrointestinal (GI) epithelium. Objective:To provide an overview of the normal distribution of ACE2 in the GI tract, altered ACE2 expression notably in coronavirus infection and its consequences. Materials and Methods: Pubmed and google scholar were searched using the key words ACE2 paired with GI tract, intestinal permeabilty, gut microbiota, inflammatory bowel disease. Results and Discussion: ACE2 is highly expressed in the ileum and colon in human being as well as in rodents. In this current situation of COVID-19 pandemic, downregulation of ACE2 has been reported due to internalization of the ACE2-virus complex within the cells. Although researches are still in infancy in this topic, altered luminal microbiota, increased intestinal permeability, higher level of inflammatory markers and deficient nutrient transport has been reported due to altered ACE2 expression. Conclusion:Altered expression of ACE2 has the possibility to hamper normal physiological function of the GI tract and might affect GI disease progression and prognosis.

LEPTOSPIROSIS IN THE CITY: A CASE REPORT

CASE: A 58 year-old undomiciled man with no medical history presented with three days of anorexia, malaise, abdominal pain, and decreased urination. Exam was notable for scleral icterus. Lab-work revealed sodium 133 mEq/L, BUN 132mg/dL, creatinine 8.82 mg/dL, platelet 64 K/uL, total bilirubin 6.4 mg/dL, direct bilirubin 5 mg/dL. Lab-work two years prior was normal. HCV antibody was reactive, urinalysis revealed microscopic hematuria, and cocaine was detected on toxicology. Abdominopelvic CT, MRCP and renal sonogram were non-pathologic. On hospital day 5 his creatinine downtrended but total bilirubin continued to rise to a peak of 11.2 mg/dL and a leukocytosis without fever developed (peak 21.2 K/uL). Ceftriaxone was started empirically and a workup of blood cultures, viral serologies, ANA, alpha-1 antitrypsin, complement, cryoglobulin, ceruloplasmin level, microsomal, smooth muscle and antimitochondrial antibodies was normal. Review of his history suggested exposure to rodents as he slept close to a dumpster. Pending Leptospirosis serology, the antibiotics were adjusted to doxycycline. At discharge, the WBC and platelet counts normalized while the bilirubin and creatinine downtrended. IgM serology for leptospira later resulted positive. IMPACT/DISCUSSION: Leptospirosis is a worldwide zoonotic disease commonly associated with moist environments, poor housing and inadequate sanitation. Rodents are important reservoirs, shedding spirochetes through urine. Human infection results from exposure to animal urine, contaminated soil or water, or infected animal tissue. Portals of entry include cuts, mucous membranes or conjunctivae. Person-toperson transmission is rare. The incubation period is 5-14 days and illness severity ranges from subclinical to life-threatening. Disease manifestations include jaundice with acute kidney failure (Weil's disease), rash, conjunctival suffusion, hyponatremia, thrombocytopenia, microscopic hematuria, myocarditis, pulmonary hemorrhage, and meningitis. A biphasic illness, the acute febrile bacteremic phase can last 2-9 days followed by a period of apparent improvement. An “immune” phase then follows characterized by development of complications, as in our patient. During this phase, leptospires are absent from blood but may appear in the urine. While human cases of leptospirosis are rarely reported in the US outside of Puerto Rico and Hawaii (in the absence of travel), there was a significant rise reported to the NYC DOH in 2021. A potential explanation is an increase in housing insecurity and disruptions to waste management as a consequence of the COVID-19 pandemic. CONCLUSION: Leptospirosis is an important consideration in at-risk populations who may unknowingly be exposed due to living conditions. Our case of unexpected Weil's disease in an urban setting underscores the importance of a thorough social history as well as timely recognition of uncommon infections as possible reversible causes of multi- organ failure in the context of a changing world climate.

Under or Absent Reporting of Light Stimuli in Testing of Anxiety-Like Behaviors in Rodents: The Need for Standardization.

Behavioral neuroscience tests such as the Light/Dark Test, the Open Field Test, the Elevated Plus Maze Test, and the Three Chamber Social Interaction Test have become both essential and widely used behavioral tests for transgenic and pre-clinical models for drug screening and testing. However, as fast as the field has evolved and the contemporaneous involvement of technology, little assessment of the literature has been done to ensure that these behavioral neuroscience tests that are crucial to pre-clinical testing have well-controlled ethological motivation by the use of lighting (i.e., Lux). In the present review paper, N = 420 manuscripts were examined from 2015 to 2019 as a sample set (i.e., n = ~20-22 publications per year) and it was found that only a meager n = 50 publications (i.e., 11.9% of the publications sampled) met the criteria for proper anxiogenic and anxiolytic Lux reported. These findings illustrate a serious concern that behavioral neuroscience papers are not being vetted properly at the journal review level and are being released into the literature and public domain making it difficult to assess the quality of the science being reported. This creates a real need for standardizing the use of Lux in all publications on behavioral neuroscience techniques within the field to ensure that contributions are meaningful, avoid unnecessary duplication, and ultimately would serve to create a more efficient process within the pre-clinical screening/testing for drugs that serve as anxiolytic compounds that would prove more useful than what prior decades of work have produced. It is suggested that improving the standardization of the use and reporting of Lux in behavioral neuroscience tests and the standardization of peer-review processes overseeing the proper documentation of these methodological approaches in manuscripts could serve to advance pre-clinical testing for effective anxiolytic drugs. This report serves to highlight this concern and proposes strategies to proactively remedy them as the field moves forward for decades to come.