RHEUMATOLOGISTS BEWARE OF LOW ALP: A CASE OF HYPOPHOSPHATASIA MISDIAGNOSED AS FIBROMYALGIA, CAUSING LONG DIAGNOSTIC DELAY

BackgroundHypophosphatasia(HPP), a rare, inherited metabolic disease featuring low serum alkaline phosphatase (ALP) activity due to ALPL (encoding tissue non-specific alkaline phosphatase) gene mutation[1,2]. A wide-ranging clinical spectrum is often seen due to defective mineralisation affecting teeth, bones, joints and muscles[1]. This disease has a prevalence of 1/6370 in Europe and is often misdiagnosed and underdiagnosed with a diagnostic delay of more than ten years[1] The treatment is often supportive for milder cases and enzyme replacement therapy in severe cases.ObjectivesTo share this case to raise awareness among Rheumatologists.MethodsThis 58-year-old Caucasian female had her first HPP symptom as early eruption of deciduous teeth, along with recurrent dental infections and gum problems. She was diagnosed with flat feet at age five, had a big toe fracture at sixteen, followed by a metatarsal fracture. She experienced leg muscle cramps and aches, affecting her performance in sport during school life.At the age of thirty she began noticing weakness in arms and legs, which progressed over the years. She faced significant early morning stiffness along with painful ribs, hips, knees, shoulders, and small joints of feet when walking.She was diagnosed with Fibromyalgia at the age of forty-four. The following ten years she met numerous specialists including rheumatologist, pain specialist and physiotherapists. She was also diagnosed with early osteoarthritis, pernicious anaemia, hyperlipidemia, functional neurological syndrome, and central sensitization syndrome. She had multiple trials of steroids and opioids, all of which were stopped either due to side effects or inefficiency.A major flare of symptoms five years ago rendered her bedbound for three months, following which a chemical pathologist noticed a persistent low ALP levels and decided to investigate for HPP. It took another four years to complete these investigations due to the coronavirus pandemic.Currently, she is unable to weight bear or climb stairs and must stay indoors or in bed during flareup. She moved into a ground floor flat at the age of 54 and use a walking stick occasionally. By 58, she is unable to work and had given up her own business due to pain, weakness, and disability.ResultsOn clinical assessment, her height is 160 cm, faced difficulty getting up from chair, has an antalgic waddling gait, with a 6-minute walking distance of 60 metre, stopped after three minutes, and had a Brief Pain Inventory pain severity score of 7/10. Her ALP level is 24 U/L and PLP/PA ratio is 18.8 (ref < 5), and genetic testing showed heterozygous missense variant of ALPL gene mutation.ConclusionIt took more than forty years to reach a conclusive diagnosis of childhood onset HPP. Low ALP level is a signature of HPP and warrants investigations. Diagnosis can be challenging due to the rareness and variable presentation, however recognition of HPP features is crucial for timely referral, optimal disease management and potential improvement in quality of life.References[1]Högler W, Langman C, Gomes da Silva H, Fang S, Linglart A, Ozono K, Petryk A, Rockman-Greenberg C, Seefried L, Kishnani PS. Diagnostic delay is common among patients with hypophosphatasia: initial findings from a longitudinal, prospective, global registry. BMC Musculoskelet Disord. 2019 Feb 14;20(1):80. doi:10.1186/s12891-019- 2420-8. PMID: 30764793;PMCID: PMC6376686.[2]Injean P, Lee S, Downey C. Hypophosphatasia May Be Misdiagnosed as Fibromyalgia: A Single Center Experience []. Arthritis Rheumatol. 2020;72 (suppl 10). https://acrs.org//hypophosphatasia-may-be-misdiagnosed-as- ibromyalgia-a-single-center-experience/. Accessed January 14, 2023.[3]Lefever E, Witters P, Gielen E, Vanclooster A, Meersseman W, Morava E, Cassiman D, Laurent MR. Hypophosphatasia in Adults: Clinical Spectrum and Its Association With Genetics and Metabolic Substrates. J Clin Densitom. 2020 Jul-Sep;23(3):340- 48. doi: 10.1016/j.jocd.2018.12.006. Epub 2018 Dec 21. PMID: 30655187.Acknowledgements:N L.Disclosure of InterestsNone Declared.

Molecular Docking Based in-Silico Approach Reveals the Potential Inhibitory Activity of Nsaids against Sars-Cov-2 Proteins

The rapid rate of mutation of the RNA genome of the Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for the emergence of viral variants, leading to the enhanced survivability of the virus. Hence, searching for new drugs that can restrict new viral infections by interacting with wild-type and mutated viral proteins is important. However, new drug development's economic and time-constraining nature makes drug repurposing a more viable solution to address the problem. In this work, we conducted a computational study to screen 23 Non-Steroidal Anti-Inflammatory Drugs (NSAID) interactions with 5 major viral proteins of SARS-CoV-2 that are mainly involved in host infection. Our in-silico results establish a database that shows that different NSAID ligands interact with the different viral proteins with good binding affinities. Stabilizing point mutations were introduced within the conserved amino acids involved in ligand-protein interactions. Redocking the NSAID ligands with these mutated viral proteins showed that the NSAID ligands could bind with the mutated and wild-type viral proteins with comparable binding affinities. We conclude that the NSAID ligands could be repurposed as therapeutic drugs against the SARS-CoV-2 virus. Additionally, our work generated a repository that includes binding affinities, possible modes of interaction, and specific interacting residues of the protein (wild-type and mutated) ligand complexes that could be used for future validation studies. Further, our results point to the potential of these drugs to treat other viral infections with similar disease etiology.Copyright All © 2023 are reserved by International Journal of Pharmaceutical Sciences and Research.

Respiratory Syncytial Virus (RSV) infections in children: recent concepts in immunopathogenesis and diagnostic dilemma

Respiratory viral infections are important cause of morbidity and mortality in early life. The relative influence of host and viral factors possibly contribute to the disease pathogenesis. Predisposing conditions like prematurity, Low birth weight and congenital heart diseases etc. have been incriminated in the disease progression. The development of cough, wheezing, and tachypnea, usually peaking on days 4 to 5, go parallel with host cytokine responses and viral load. Various host cytokines, chemokines and molecules involved in the immune response against RSV infection might be responsible for the outcome of the disease process. Nasopharyngeal aspirates (NPAs) from children (n = 349) between 2013-2017 were subjected for IL-17A, IFN-gamma, TNF-alpha, IL-10, IL-6 levels by CBA and MMP-9 and TIMP-1 levels by ELISA. The viral load in RSV positive samples and cytokine levels were correlated with the WHO criteria for acute lower respiratory tract illness (ALRTI). RSV viral load, Pro-inflammatory cytokine (TNF-alpha) levels in severe ALRTI patients were significantly higher than the ALRTI patients [p<0.001]. Whereas Th17 cytokine (IL-17) was found to be significantly higher (p<0.05) in ALRTI patients than severe patients. MMP-9 is secreted in higher levels in severe ALRTI patients (n = 77) in comparison to Acute LRTI patients (n = 35) with an increase of thirty seven fold (p<0.001). Thus, the study highlights the role of TNF -alpha, IL-17 and Th2 cytokine biasness in the pathogenesis of RSV disease with the possible contribution of higher MMP-9/TIMP-1 ratio as a bad prognostic marker towards disease severity. To study the gene expression of autophagy and mTOR signalling pathways in RSV infected children with ALRTI. Nasopharyngeal aspirate (NPA) samples (n = 145) from children suffering from ALRTI were subjected for detection of RSV (Oct 2019 to March 2020). Semi-quantitative gene expression analysis for 5 representative genes each of mTOR signalling and autophagy pathway were performed in respiratory tract epithelial cells using 25 RSV positive cases and 10 healthy controls subjects. Autophagy gene expression analysis revealed significant upregulation in NPC1 and ATG3 autophagy genes. mTOR, AKT1 and TSC1 genes of mTOR pathway were significantly down-regulated in RSV positive patients except RICTOR gene which was significantly upregulated. Thus, survival of RSV within autophagosome might have been facilitated by upregulation of autophagy and downregulation of mTOR signalling genes. To assess the impact of SARS-CoV2 pandemic on RSV, samples were collected from children with ALRTIs admitted to emergency, PICU and indoor admissions during pre-pandemic period (October 2019 to February 2020;n = 166) and during COVID-19 Pandemic (July 2021 to July 2022;n = 189, SARS-CoV2 negative). These NP swabs were analyzed for pdm InfA H1N1, InfA H3N2, Inf B, RSV, hMPV, hBoV, hRV, PIV-2 and PIV-3 by PCR. Higher proportion of children with ALRTIs have had virus/es isolated during pre-pandemic period than during pandemic period (p<0.001). During pre-pandemic period, significantly higher proportion of children had RSV positivity (p<0.001);and significantly lower positivity for hRV (p<0.05), hMPV (p<0.05), and hBoV (p <= 0.005). The occurrence of COVID-19 pandemic has significantly impacted the frequency and pattern of detection of RSV among hospitalized children with LRTIs. RSV Fusion protein plays a critical role in the entry of the virus into the host cell by initiating the fusion of host and viral membranes. It happens to be a target of neutralizing antibodies paving the way as a vaccine candidate. Hence effort was made to introduce point mutation in hRSV fusion protein which can confer stability in its prefusion form. In-silico a stable structure of RSV fusion protein was generated making it a potential vaccine candidate. The timely diagnosis of RSV infection in this population is important for initiating therapy and instituting appropriate infection prevention measures. Serological testing is not widely used for the diagnosis of RSV. C ll Cultures including shell vial culture were used for RSV diagnosis. However, culture approaches lack sensitivity, often quite significantly, compared to nucleic acid amplification assays for the diagnosis of RSV infections. Molecular multiplex assays now offer increased sensitivity for a more accurate diagnosis. However issues with the use of these types of commercial panel assays include the requirement for substantial training, quality systems, and infrastructure to maintain and run these assays and many a times identification of viruses where the true pathogenic potential of those multiple viruses are debatable. Studies are available with laboratory- developed nucleic acid amplification test systems for the detection of RSVA and RSVB in clinical specimens either by PCRbased technologies or RT-LAMP. Gene targets of laboratory-developed molecular assays point towards M gene and the N gene in RSVA and -B with the benefits of flexibility to modify assays when targets are under evolutionary pressure to change, as well as a perceived initial low cost to carry out testing.

Current genetic strategies to investigate gene functions in Trichoderma reesei.

The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina, Ascomycota) is a well-known lignocellulolytic enzymes-producing strain in industry. To increase the fermentation titer of lignocellulolytic enzymes, random mutagenesis and rational genetic engineering in T. reesei were carried out since it was initially found in the Solomon Islands during the Second World War. Especially the continuous exploration of the underlying regulatory network during (hemi)cellulase gene expression in the post-genome era provided various strategies to develop an efficient fungal cell factory for these enzymes' production. Meanwhile, T. reesei emerges competitiveness potential as a filamentous fungal chassis to produce proteins from other species (e.g., human albumin and interferon α-2b, SARS-CoV-2 N antigen) in virtue of the excellent expression and secretion system acquired during the studies about (hemi)cellulase production. However, all the achievements in high yield of (hemi)cellulases are impossible to finish without high-efficiency genetic strategies to analyze the proper functions of those genes involved in (hemi)cellulase gene expression or secretion. Here, we in detail summarize the current strategies employed to investigate gene functions in T. reesei. These strategies are supposed to be beneficial for extending the potential of T. reesei in prospective strain engineering.

The emerging Omicron variant spike mutation: the relative receptor-binding domain affinity and molecular dynamics

This study aims to fill a knowledge gap in our understanding of Omicron variant receptor-binding domain (RBD) interactions with host cell receptor, angiotensin-converting enzyme 2 (ACE2). Protein-protein docking, scoring, and filtration were all performed using the HDOCK server. A coarse-grained prediction of the changes in binding free energy caused by point mutations in Omicron RBD was requested from the Binding Affinity Changes upon Mutation (BeAtMuSiC) tools. GROMACS was utilized to perform molecular dynamics simulations (MD). Within the 15 mutations in Omicron RBD, several mutations have been linked to increased receptor affinity, immunological evasion, and inadequate antibody response. Wild-type (wt) SARS-CoV-2 and its Omicron variant have 92.27% identity. Nonetheless, Omicron RBD mutations resulted in a slight increase in the route mean square deviations (RMSD) of the Omicron structural model during protein-protein docking, as evidenced by RMSDs of 0.47 and 0.85 A for the wt SARS-CoV-2 and Omicron RBD-ACE2 complexes, respectively. About five-point mutations had essentially an influence on binding free energy, namely G6D, S38L, N107K, E151A, and N158Y. The rest of the mutations were expected to reduce the binding affinity of Omicron RBD and ACE2. The MD simulation supports the hypothesis that Omicron RBD is more stably bound to ACE2 than wt SARS-CoV-2 RBD. Lower RMSD and greater radius of gyration (Rg) imply appropriate Omicron structure 3D folding and stability. However, the increased solvent accessible surface area (SASA) with a greater Omicron shape may have a different interaction with receptor binding and regulate virus entrance. Omicron RBD's mutations help it maintain its structural stability, compactness, ACE2 binding, and immune evasion.Copyright © 2022 AEPress, s.r.o.. All rights reserved.

Analysis of drug resistance gene in Mycoplasma pneumoniae and 13 pathogens in bronchoalveolar lavage fluid of children with Mycoplasma pneumoniae pneumonia. TT -.

Objective To investigate drug resistance gene in Mycoplasma pneumoniae (MP) and the distribution of 13 respiratory pathogens in bronchoalveolar lavage fluid(BALF) of children with Mycoplasma pneumoniae pneumonia(MPP). Methods A total of 100 BALF of children with MPP in Peking University Third Hospital and Peking University First Hospital from January 2018 to January 2019 were collected. Fluorogenic quantitative PCR was used to detect nucleic acid and it's drug resistance gene of MP and multiple PCR method was adopted to detect influenza A virus, influenza A virus - H1 N1, influenza A virus - H3 N2, influenza B, human parainfluenza virus, adenovirus, human bocavirus, human rhino virus, Chlamydia pneumoniae, human metapneumovirus, MP, human corona virus, and respiratory syncytial virus gene, and the results were compared by using Chi square test. Results In 100 BALF samples, MP and drug resistance gene were detected by fluorogenic quantitative PCR. Totally, 83 cases (83. 00%) were MP positive and 78 cases (93. 98%) were drug resistant. All of them had the point mutations A2063G in V region of 23S rRNA domain. A total of 13 kinds of respiratory pathogens were detected by multiplex PCR method, and 89 cases (89. 00%) were positive. Totally, 79 cases (79. 00%) were MP positive, of which 74 cases (74. 00%) detected only MP, and 5 cases (5. 00%) detected MP combined with other pathogens. Other pathogens were detected in 10 cases (10. 00%). The virus detection rate of 0-4 years old group was higher than that of > 4-6 years old group (P - 0. 042) and > 6 years old group (P =0. 002), and the differences were statistically significant. Conclusions MP can be detected in most BALF samples of MPP children, the drug resistance phenomenon is serious, and the main point mutation is A2063G. There were other respiratory pathogens and 2 or 3 pathogens were detected in a small number of BALF samples.Copyright © 2022 Authors. All rights reserved.

5th ECOP, European Conference of Oncology Pharmacy

The proceedings contain 130 papers. The topics discussed include: frequency of measuring body weight in (sub)populations of patients with cancer treated with chemotherapy;simple approach to enhance green tea epigallocatechin gallate stability in aqueous solutions and it bioavailability: experimental and theoretical approaches;incidence of cisplatin-induced nephrotoxicity and associated risk factors: single-center experience;impact of the 2019 coronavirus pandemic on cancer treatment in the Calabria Region, Italy;Palbociclib associated neutropenia in clinical practice;successful introduction of a point mutation into the genome of a primary colon cancer cell line using CRISPR base editing technology;incidence of cisplatin-induced nephrotoxicity and associated risk factors: single-center experience;real world data of alk-inhibitors in patients with advanced or metastic non-small cell lung cancer;pembrolizumab in non-small-cell lung cancer: a systematic review of real life data in Spain;gynecomastia in a male after imatinib treatment for chronic myeloid leukemia;and results after discontinuation of pembrolizumab in metastatic melanoma or lung cancer patients: real-word experience.

Mutation-driven parallel evolution in emergence of ACE2-utilizing sarbecoviruses.

Mutation and recombination are two major genetic mechanisms that drive the evolution of viruses. They both exert an interplay during virus evolution, in which mutations provide a first ancestral source of genetic diversity for subsequent recombination. Sarbecoviruses are a group of evolutionarily related ß-coronaviruses including human severe acute respiratory syndrome coronavirus (SARS-CoV) and SARS-CoV-2 and a trove of related animal viruses called SARS-like CoVs (SL-CoVs). This group of members either use or not use angiotensin-converting enzyme 2 (ACE2) as their entry receptor, which has been linked to the properties of their spike protein receptor binding domains (RBDs). This raises an outstanding question regarding how ACE2 binding originated within sarbecoviruses. Using a combination of analyses of phylogenies, ancestral sequences, structures, functions and molecular dynamics, we provide evidence in favor of an evolutionary scenario, in which three distinct ancestral RBDs independently developed the ACE2 binding trait via parallel amino acid mutations. In this process, evolutionary intermediate RBDs might be firstly formed through loop extensions to offer key functional residues accompanying point mutations to remove energetically unfavorable interactions and to change the dynamics of the functional loops, all required for ACE2 binding. Subsequent optimization in the context of evolutionary intermediates led to the independent emergence of ACE2-binding RBDs in the SARS-CoV and SARS-CoV-2 clades of Asian origin and the clade comprising SL-CoVs of European and African descent. These findings will help enhance our understanding of mutation-driven evolution of sarbecoviruses in their early history.

High-Affinity Antibodies Designing of SARS-CoV-2 Based on Molecular Dynamics Simulations.

SARS-CoV-2 has led to a global pandemic of new crown pneumonia, which has had a tremendous impact on human society. Antibody drug therapy is one of the most effective way of combating SARS-CoV-2. In order to design potential antibody drugs with high affinity, we used antibody S309 from patients with SARS-CoV as the target antibody and RBD of S protein as the target antigen. Systems with RBD glycosylated and non-glycosylated were constructed to study the influence of glycosylation. From the results of molecular dynamics simulations, the steric effects of glycans on the surface of RBD plays a role of "wedge", which makes the L335-E340 region of RBD close to the CDR3 region of the heavy chain of antibody and increases the contact area between antigen and antibody. By mutating the key residues of antibody at the interaction interface, we found that the binding affinities of antibody mutants G103A, P28W and Y100W were all stronger than that of the wild-type, especially for the G103A mutant. G103A significantly reduces the distance between the binding region of L335-K356 in the antigen and P28-Y32 of heavy chain in the antibody through structural transition. Taken together, the antibody design method described in this work can provide theoretical guidance and a time-saving method for antibody drug design.

A Probabilistic Approach to Evaluate the Likelihood of Artificial Genetic Modification and Its Application to SARS-CoV-2 Omicron Variant

A method to find a probability that a given bias of mutations occur naturally is proposed to test whether a newly detected virus is a product of natural evolution or a product of non-natural process such as genetic manipulation. The probability is calculated based on the neutral theory of molecular evolution and binominal distribution of non-synonymous (N) and synonymous (S) mutations. Though most of the conventional analyses, including dN/dS analysis, assume that any kinds of point mutations from a nucleotide to another nucleotide occurs with the same probability, the proposed model takes into account the bias in mutations, where the equilibrium of mutations is considered to estimate the probability of each mutation. The proposed method is applied to evaluate whether the Omicron variant strain of SARS-CoV-2, whose spike protein includes 29 N mutations and only one S mutation, can emerge through natural evolution. The result of binomial test based on the proposed model shows that the bias of N/S mutations in the Omicron spike can occur with a probability of 2.0 × 10−3 or less. Even with the conventional model where the probabilities of any kinds of mutations are all equal, the strong N/S mutation bias in the Omicron spike can occur with a probability of 3.7 × 10−3, which means that the Omicron variant is highly likely a product of non-natural process including artifact. © 2022 Information Processing Society of Japan.

Engineering a Pseudotyped Lentiviral Platform to Enable Lineage-Specific Transduction of Immune Cells

Background Cell-specific transduction remains one of the next frontiers for virally-delivered gene therapy. Efforts to achieve cell-specific transduction have largely been limited to borrowing of preexisting viral glycoproteins and pseudotyping viral surface envelopes to result in altered tropism. VSVG is derived from vesicular stomatitis virus (VSV) and is one of the most commonly used lentiviral (LV) pseudotype glycoproteins as its cognate receptor (LDLR) is present on nearly all dividing and non-dividing cells, enabling broad tropism of VSVG-pseudotyped LVs. Methods Our lab recently developed a receptor-blinded version of VSVG, in which point mutations (K47Q, R354A) of this glycoprotein results in a mutated VSVG with inability to bind and infect LDLR-expressing cells. This mutant viral glycoprotein, which we call VSVGmut, thereby loses its broad tropism, but critically retains its fusogen ability, enabling codisplay of a new LV pseudotype ligand to drive LV tropism. Results Initial experiments displaying high-affinity anti-CD19 scFvs alongside VSVGmut on the LV surface demonstrated infection of CD19+, but not CD19- cells. Subsequent work using endogenous ligands (CD80), Fabs (a-CD3e), cytokines (IL-13), viral glycoproteins (SARS-CoV-2 RBD), and peptide MHCs (pMHCs) revealed the modularity of this platform for achieving potent transduction of on-target cells, with minimal infection of bystander cells, across a range of affinities (pM to uM) and at frequencies as low as 1 in 100,000. This technology allowed for library on library screening of 96 viral pMHC-displaying LVs against a repertoire of >450,000 TCRs in pool, which accurately uncovered EBV- and Flu-specific TCRs through scRNA sequencing. Conclusions The VSVGmut platform has resulted in our lab's ability to pair pMHCs with cognate TCRs and viral surface antigens with cognate BCRs, in addition to achieving lineagespecific transduction of T and B cell subsets, setting the stage for cell-specific gene therapy.

Surface charge changes in spike RBD mutations of SARS-CoV-2 and its variant strains alter the virus evasiveness via HSPGs: A review and mechanistic hypothesis.

With the COVID-19 pandemic continuing, more contagious SARS-CoV-2 variants, including Omicron, have been emerging. The mutations, especially those that occurred on the spike (S) protein receptor-binding domain (RBD), are of significant concern due to their potential capacity to increase viral infectivity, virulence, and breakthrough antibodies' protection. However, the molecular mechanism involved in the pathophysiological change of SARS-CoV-2 mutations remains poorly understood. Here, we summarized 21 RBD mutations and their human angiotensin-converting enzyme 2 (hACE2) and/or neutralizing antibodies' binding characteristics. We found that most RBD mutations, which could increase surface positive charge or polarity, enhanced their hACE2 binding affinity and immune evasion. Based on the dependence of electrostatic interaction of the epitope residue of virus and docking protein (like virus receptors or antibodies) for its invasion, we postulated that the charge and/or polarity changes of novel mutations on the RBD domain of S protein could affect its affinity for the hACE2 and antibodies. Thus, we modeled mutant S trimers and RBD-hACE2 complexes and calculated their electrotactic distribution to study surface charge changes. Meanwhile, we emphasized that heparan sulfate proteoglycans (HSPGs) might play an important role in the hACE2-mediated entry of SARS-CoV-2 into cells. Those hypotheses provide some hints on how SARS-CoV-2 mutations enhance viral fitness and immune evasion, which may indicate potential ways for drug design, next-generation vaccine development, and antibody therapies.

Implementing a database to track patients’ compliance in the high-risk pancreatic cancer clinic

BackgroundPancreatic cancer is projected to become the second leading cause of cancer death in the United States by 2030. Screening of high-risk individuals (HRI’s) using endoscopic ultrasound (EUS) or magnetic resonance imaging (MRI), specifically magnetic resonance cholangiopancreatography (MRCP), saves lives and reduces morbidity and mortality. Screening can detect surgically resectable lesions, which is key to survival since most lesions present as unresectable or metastatic. The specific aims of this project were to address the quality improvement process to track adherence of the patients who participate in the high-risk pancreatic cancer clinic at UMass Memorial Healthcare.MethodsPatients deemed to be high risk according to the Cancer of the Pancreas Screening (CAPS) Consortium Guidelines included having at least one first-degree relative with a diagnosis of pancreatic cancer, a genetic mutation that places them at higher risk for pancreatic cancer, or both are the cohort. The data was collected via EMR using REDCap, a secure web application to analyze high-risk patients’ adherence with yearly screening. The method utilized a quality improvement project design.ResultsResults demonstrated a majority of participants were female, 53% had a first-degree relative with pancreatic cancer, a majority of participants did not carry a genetic mutation while the highest mutation finding included 16 participants (14.4%) with a BRCA 2 gene mutation. A majority of participants, 64.9% (n=72) were non-compliant with screening while 35% (n=39) had yearly screening. Participants who were non-adherent with screening endorsed multiple factors for non-adherence including not believing surveillance was necessary, not qualifying for screening at the time of initial appointment, not receiving reminder phone calls for clinic appointments and surveillance imaging, other health concerns, high insurance deductibles, the COVID pandemic, and high anxiety.ConclusionsMitigation of the reasons for non-adherence is a crucial practice implication. Patient education regarding the importance of long-term yearly screening and teaching patients self-efficacy behaviors may improve screening adherence. Prospectively analyzing compliance will reduce missed care opportunities. Health care policies must be amended to require insurance companies to cover the screening of HRI’s.

Molecular diagnostic for SARS-CoV-2: an experimental External Quality Assessment Scheme

Introduction: external Quality Assessment (EQA) is a valuable tool to monitor and improve the analytical performances of clinical laboratories. To guarantee suitable results also during the COVID-19 pandemic, EQA scheme providers have implemented specific schemes assessing different SARS-CoV-2 diagnostic systems. This study aims to describe the results collected in an experimental EQA scheme for molecular diagnostic of SARS-CoV-2 managed by INSTAND eV with the collaboration of the Centre of Biomedical Research for Quality in Laboratory Medicine for Veneto Region Laboratories. Methods: the qualitative EQA results collected in three surveys (two in 2020 and one in 2021) for 18 samples total, have been summarized to identify the percentage of laboratory results per sample. Control samples were provided by NationalesKonsiliarlaboratorium fur Coronaviren of Berlin. Results: even though the average of the participating laboratories strongly decreased between surveys, a good agreement was found among results (95% to 99.8%). A totally of 0.2% - 4% of incorrect results and 0% - 1.1% of indeterminate results were reported. In addition, the sequencing analysis and the point mutations analysis, included in the last analyzed survey, revealed a good agreement between participating laboratories with an overall score from 74.8% to 89.6% for the sequencing and from 90.65% to 95.33% for the point mutations, respectively. Conclusions: the EQA programs are a fundamental quality assurance tool to evaluate the laboratory performance and to appreciate the State-of-the-Art of the different diagnostic systems used by participating laboratories. The need for an EQA scheme for every test performed in the laboratory is mandatory to guarantee patient safety.

Comparison of Insertion, Deletion, and Point Mutations in the Genomes of Human Adenovirus HAdvC-2 and SARS-CoV-2.

Virus genome mutation profiles with insertion, deletion, and point mutations have recently been revealed to differ remarkably between viruses. In RNA viruses like human coronaviruses or influenza viruses, genome samples collected over two to three decades usually show point mutations in 10-20% of the bases, while the rate of insertion and/or deletion mutations (indels) largely depends on the virus. This study evaluates the mutation profiles of DNA viruses by comparing a recently sampled genome of human adenovirus species C type 2 (isolate SG06/HAdvC2/2016) with a genome of the same species sampled in the 1970s. It was found insertions of 23 bases at seven sites and deletions of 22 bases at nine sites. The longest indels were 6-base insertions in E2B and L4. All indels in the coding regions were in-frame mutations with base lengths in multiples of three. In the non-coding regions, the lengths of the indels ranged from 1-4 consecutive bases. Long indels with more than 10 consecutive bases, which comprise nearly half of indels in the SARS-CoV-2 genome, were absent. In other sites, the point mutation rate was approximately 0.3%, which was significantly lower than in RNA viruses. In summary, the estimated point mutation rate in human adenovirus species C type 2 (HAdvC-2) was over 10 times lower than in RNA viruses. Unlike the relatively long indels in the SARS-CoV-2 genome, the indels in HAdvC-2 were short, with 6 or fewer consecutive bases.

The impacts of 13 novel mutations of SARS-CoV-2 on protein dynamics: In silico analysis from Turkey

SARS-CoV-2 inherits a high rate of mutations making it better suited to the host since its fundamental role in evolution is to provide diversity into the genome. This research aims to identify variations in Turkish isolates and predict their impacts on proteins. To identify novel variations and predict their impacts on protein dynamics, in silico methodology was used. The 411 sequences from Turkey were analysed. Secondary structure prediction by Garnier-Osguthorpe-Robson (GOR) was used. To find the effects of identified Spike mutations on protein dynamics, the SARS-CoV-2 structures (PDB:6VYB, 6M0J) were uploaded and predicted by Cutoff Scanning Matrix (mCSM), DynaMut and MutaBind2. To understand the effects of these mutations on Spike protein molecular dynamics (MD) simulation was employed. Turkish sequences were aligned with sequences worldwide by MUSCLE, and phylogenetic analysis was performed via MegaX. The 13 novel mutations were identified, and six of them belong to spike glycoprotein. Ten of these variations revealed alteration in the secondary structure of the protein. Differences of free energy between the reference sequence and six mutants were found below zero for each of six isolates, demonstrating these variations have stabilizing effects on protein structure. Differences in vibrational entropy calculation revealed that three variants have rigidification, while the other three have a flexibility effect. MD simulation revealed that point mutations in spike glycoprotein and RBD:ACE-2 complex cause changes in protein dynamics compared to the wild-type, suggesting possible alterations in binding affinity. The phylogenetic analysis showed Turkish sequences distributed throughout the tree, revealing multiple entrances to Turkey.

Minor Changes with Large Implications: Modeling Amino Acid Mutations in SARSCoV Monoclonal Antibodies (80R and 362) Towards the Design of More Universal Antibodies

The SARS-CoV-2 virus is responsible for the COVID-19 pandemic which continues to impact nearly every person on Earth, having caused over 1.8 million deaths. Two anti-SARS-CoV monoclonal antibodies (MAbs) 80R and 362 are known to bind to epitopes on the spike protein receptor-binding domain (RBD) and neutralize the virus. To investigate this further and hypothesize structures for potentially more effective antibodies, undergraduate students cooperated in teams as part of the CREST (Connecting Researchers, Educators, and STudents) Program with the Center for Biological Modeling. Working collaboratively, students from eight universities nationwide applied their knowledge to build 3-D printed models to explain a particular protein-based molecular story using crystal structures of proteins described in the literature. The Nova Southeastern University (NSU) CREST team modeled and compared the 80R antibody that binds to SARS-CoV-1 and the MAb362 antibody that can bind to both SARS-CoV-1 and SARS-CoV-2. Students developed skills with protein visualization software including Pymol and Jmol to design models which showed the 80R and 362 antibodies binding to the RBD of the corresponding proteins. By studying the point mutation differences between the two antibodies (80R and 362), a potentially more universal antibody (named NSU1 in this study) was modeled. This hypothesized antibody was expected to bind more effectively to future mutations in the SARS spike protein. At the binding interface between these antibodies and the SARS spike protein, MAb362 mutations trend smaller and less polar including: Arg149Ser, Asn151Ser, Asp170Gly, and Trp213Ser. Due to the trend of smaller amino acids appearing in the MAb362 binding interface, it was hypothesized that more space in this area could allow antibodies to be more resistant to future SARS-CoV spike protein structure variations. NSU1 was modeled based on MAb362 with the following four additional mutations: Asp103Gly, Trp104Leu, Gly170Ser, and Arg211Val. All of these except for Gly170 are mutations that decreased size and polarity of amino acid residues within the binding interface. Position 170 is Asp on the 80R structure and thus a mutation to Ser is still expected to maintain this trend of smaller residues in the antibody. Due to the additional space created due to these amino acid substitutions in the binding region between the antibody and RBD of the spike protein, NSU1 was predicted to be more resistant to spike protein mutations. These models allowed for deeper understanding of the impact that mutations in antibodies can have on binding interactions with viral proteins. Additionally, the modeling process also provided insight into the molecular structure of a potentially more universal antibody against variations in SARS-CoV.

How Do Point Mutations Enhancing the Basic Character of the RBDs of SARS-CoV-2 Variants Affect Their Transmissibility and Infectivity Capacities?

The spread of SARS-CoV-2 variants in the population depends on their ability to anchor the ACE2 receptor in the host cells. Differences in the electrostatic potentials of the spike protein RBD (electropositive/basic) and ACE2 receptor (electronegative/acidic) play a key role in both the rapprochement and the recognition of the coronavirus by the cell receptors. Accordingly, point mutations that result in an increase in electropositively charged residues, e.g., arginine and lysine, especially in the RBD of spike proteins in the SARS-CoV-2 variants, could contribute to their spreading capacity by favoring their recognition by the electronegatively charged ACE2 receptors. All SARS-CoV-2 variants that have been recognized as being highly transmissible, such as the kappa (κ), delta (δ) and omicron (o) variants, which display an enhanced electropositive character in their RBDs associated with a higher number of lysine- or arginine-generating point mutations. Lysine and arginine residues also participate in the enhanced RBD-ACE2 binding affinity of the omicron variant, by creating additional salt bridges with aspartic and glutamic acid residues from ACE2. However, the effects of lysine- and arginine-generating point mutations on infectivity is more contrasted, since the overall binding affinity of omicron RBD for ACE2 apparently results from some epistasis among the whole set of point mutations.

Ultrasensitive Electrochemical Detection of Mutated Viral RNAs with Single-Nucleotide Resolution Using a Nanoporous Electrode Array (NPEA).

The detection of nucleic acids and their mutation derivatives is vital for biomedical science and applications. Although many nucleic acid biosensors have been developed, they often require pretreatment processes, such as target amplification and tagging probes to nucleic acids. Moreover, current biosensors typically cannot detect sequence-specific mutations in the targeted nucleic acids. To address the above problems, herein, we developed an electrochemical nanobiosensing system using a phenomenon comprising metal ion intercalation into the targeted mismatched double-stranded nucleic acids and a homogeneous Au nanoporous electrode array (Au NPEA) to obtain (i) sensitive detection of viral RNA without conventional tagging and amplifying processes, (ii) determination of viral mutation occurrence in a simple detection manner, and (iii) multiplexed detection of several RNA targets simultaneously. As a proof-of-concept demonstration, a SARS-CoV-2 viral RNA and its mutation derivative were used in this study. Our developed nanobiosensor exhibited highly sensitive detection of SARS-CoV-2 RNA (∼1 fM detection limit) without tagging and amplifying steps. In addition, a single point mutation of SARS-CoV-2 RNA was detected in a one-step analysis. Furthermore, multiplexed detection of several SARS-CoV-2 RNAs was successfully demonstrated using a single chip with four combinatorial NPEAs generated by a 3D printing technique. Collectively, our developed nanobiosensor provides a promising platform technology capable of detecting various nucleic acids and their mutation derivatives in highly sensitive, simple, and time-effective manners for point-of-care biosensing.