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Background Effective inhibition of thrombosis without generating bleeding risks is a major challenge in medicine. Accumulating evidence suggests that this can be achieved by inhibition of coagulation factor XII (FXII), as either its knock-out or inhibition in animal models efficiently reduced thrombosis without affecting normal hemostasis. Based on these findings, highly specific inhibitors for human FXII(a) are under development. However, currently, in vivo studies on their efficacy and safety are impeded by the lack of an optimized animal model expressing the specific target, that is, human FXII. Objective The primary objective of this study is to develop and functionally characterize a humanized FXII mouse model. Methods A humanized FXII mouse model was generated by replacing the murine with the human F12 gene (genetic knock-in) and tested it in in vitro coagulation assays and in in vivo thrombosis models. Results These hF12\(^{KI}\) mice were indistinguishable from wild-type mice in all tested assays of coagulation and platelet function in vitro and in vivo, except for reduced expression levels of hFXII compared to human plasma. Targeting FXII by the anti-human FXIIa antibody 3F7 increased activated partial thromboplastin time dose-dependently and protected hF12\(^{KI}\) mice in an arterial thrombosis model without affecting bleeding times. Conclusion These data establish the newly generated hF12\(^{KI}\) mouse as a powerful and unique model system for in vivo studies on anti-FXII(a) biologics, supporting the development of efficient and safe human FXII(a) inhibitors.

Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational attributes of the molecular complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike protein. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provides structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.

Megakaryocytes are large cells in the bone marrow that give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of the mature cells in close proximity to bone marrow sinusoids, and the formation of protrusions, which are elongated and shed within the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics, were not affected in the absence of RhoB. However, in vitro–generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with reduced levels of acetylated α-tubulin. Our findings imply that the reduction of this tubulin posttranslational modification results in impaired microtubule dynamics, which might contribute to microthrombocytopenia in RhoB-deficient mice. Importantly, we demonstrate that RhoA and RhoB are localized differently and have selective, nonredundant functions in the megakaryocyte lineage.

Megakaryocytes are large cells in the bone marrow, which give rise to blood platelets. Platelet biogenesis involves megakaryocyte maturation, the localization of mature cells in close proximity to bone marrow sinusoids and the formation of protrusions, which are shed into the circulation. Rho GTPases play important roles in platelet biogenesis and function. RhoA-deficient mice display macrothrombocytopenia and a striking mislocalization of megakaryocytes into bone marrow sinusoids and a specific defect in G-protein signaling in platelets. However, the role of the closely related protein RhoB in megakaryocytes or platelets remains unknown. In this study, we show that, in contrast to RhoA deficiency, genetic ablation of RhoB in mice results in microthrombocytopenia (decreased platelet count and size). RhoB-deficient platelets displayed mild functional defects predominantly upon induction of the collagen/glycoprotein VI pathway. Megakaryocyte maturation and localization within the bone marrow, as well as actin dynamics were not affected in the absence of RhoB. However, in vitro generated proplatelets revealed pronouncedly impaired microtubule organization. Furthermore, RhoB-deficient platelets and megakaryocytes displayed selective defects in microtubule dynamics/stability, correlating with pronouncedly reduced levels of acetylated α-tubulin. Our findings imply that absence of this tubulin posttranslational modification results in decreased microtubule stability leading to microthrombocytopenia in RhoB-deficient mice. Our data thus points to specifically impaired microtubule - but not actin - dynamics as a general mechanism underlying the manifestation of microthrombocytopenia in vivo. We furthermore demonstrate that RhoA and RhoB have specific, non-redundant functions in the megakaryocyte lineage.KEY POINTSRhoB-deficient mice display microthrombocytopeniaRhoB has different functions in the megakaryocyte lineage than RhoA and regulates microtubule dynamics

In 2020, the coronavirus disease 2019 (COVID-19) pandemic limited musculoskeletal care to urgent or “nonelective” office visits and procedures. No guidelines exist to inform patients or physicians what meets these criteria. The purpose of this multi-institutional study was to describe the differences in perceptions of urgency for musculoskeletal complaints between patients and providers during the COVID-19 pandemic. An anonymous survey was distributed to patients who visited the authors' orthopedic clinics in January and February 2020 and practicing orthopedic surgeons. The surveys were administered in May 2020 after COVID-19 was officially labeled a pandemic and included questions regarding demographic information and perceptions of orthopedic urgency. A total of 1491 patients and 128 physicians completed the surveys. A significantly higher percentage of physicians considered the following diagnoses an appropriate indication for an urgent visit compared with patients: fracture ( P P P P P P P P P P P =1.00), joint/extremity deformity without pain ( P =.113), and loss of range of motion of a joint ( P =.467). Younger patients and those with higher levels of education were significantly more likely to consider their conditions urgent. Patients may require additional education to prevent delay in treatment of urgent conditions—especially time-sensitive conditions such as neurologic compromise, tumors, and infections—when access to physicians is limited. [ Orthopedics . 2021;44(4):e534–e538.]

Entry of SARS-CoV-2, etiological agent of COVID-19, in the host cell is driven by the interaction of its spike protein with human ACE2 receptor and a serine protease, TMPRSS2. Although complex between SARS-CoV-2 spike protein and ACE2 has been structurally resolved, the molecular details of the SARS-CoV-2 and TMPRSS2 complex are still elusive. TMPRSS2 is responsible for priming of the viral spike protein that entails cleavage of the spike protein at two potential sites, Arg685/Ser686 and Arg815/Ser816. The present study aims to investigate the conformational details of complex between TMPRSS2 and SARS-CoV-2 spike protein, in order to discern the finer details of the priming of viral spike and to point candidate drug targets. Briefly, full length structural model of TMPRSS2 was developed and docked against the resolved structure of SARS-CoV-2 spike protein with directional restraints of both cleavage sites. The docking simulations showed that TMPRSS2 interacts with the two different loops of SARS-CoV-2 spike protein, each containing different cleavage sites. Key functional residues of TMPRSS2 (His296, Ser441 and Ser460) were found to interact with immediate flanking residues of cleavage sites of SARS-CoV-2 spike protein. Compared to the N-terminal cleavage site (Arg685/Ser686), TMPRSS2 region that interact with C-terminal cleavage site (Arg815/Ser816) of the SARS-CoV-2 spike protein was predicted as relatively more druggable. In summary, the present study provide structural characteristics of molecular complex between human TMPRSS2 and SARS-CoV-2 spike protein and points to the candidate drug targets that could further be exploited to direct structure base drug designing.

The recent pandemic of COVID-19, caused by SARS-CoV-2, is unarguably the most fearsome compared with the earlier outbreaks caused by other coronaviruses, SARS-CoV and MERS-CoV. Human ACE2 is now established as a receptor for the SARS-CoV-2 spike protein. Where variations in the viral spike protein, in turn, lead to the cross-species transmission of the virus, genetic variations in the host receptor ACE2 may also contribute to the susceptibility and/or resistance against the viral infection. This study aims to explore the binding of the proteins encoded by different human ACE2 allelic variants with SARS-CoV-2 spike protein. Briefly, coding variants of ACE2 corresponding to the reported binding sites for its attachment with coronavirus spike protein were selected and molecular models of these variants were constructed by homology modeling. The models were then superimposed over the native ACE2 and ACE2-spike protein complex, to observe structural changes in the ACE2 variants and their intermolecular interactions with SARS-CoV-2 spike protein, respectively. Despite strong overall structural similarities, the spatial orientation of the key interacting residues varies in the ACE2 variants compared with the wild-type molecule. Most ACE2 variants showed a similar binding affinity for SARS-CoV-2 spike protein as observed in the complex structure of wild-type ACE2 and SARS-CoV-2 spike protein. However, ACE2 alleles, rs73635825 (S19P) and rs143936283 (E329G) showed noticeable variations in their intermolecular interactions with the viral spike protein. In summary, our data provide a structural basis of potential resistance against SARS-CoV-2 infection driven by ACE2 allelic variants.

Gout is one of the oldest known diseases and described in Unani system of medicine under the term Niqras.Niqris (gout) is a type of Waja' al-Mafasil(arthritis) which is characterized by recurrent attack of acute pain and swelling primarily affecting one joint usually the metatarsal joint of big toe and small joints of hand and feet. It is a commonest joint pain creating problems in day to day life of various people of the world. In Modern drugs used for subsiding acute attacks or lowering serum uric acid are associated with potent adverse effects. Moreover, these commonly used therapeutic agents often, and for various reasons, do not achieve the desired lowering of serum urate levels to below 6.0 mg/dl. The objective of present study was to ascertain the Unani concept described in Unani classical literatures regarding various causes, symptoms and management of this common arthritic disorder by Renowned Unani Physicians with an aim to spread the knowledge for preventive measures, home remedies, regimenal therapy to get relief from the disease and the management of gout by Unani compound formulations, which are not only easily available but also have no side effect on human body. In this regard, we may conclude that spread of knowledge of enormous effective Unani components and general principles of treatment, which are being used by Unani physicians since ancient times, shall be extremely effective in the management of this musculo skeletal disorder.

Objective— It is known that both platelets and coagulation strongly influence infarct progression after ischemic stroke, but the mechanisms and their interplay are unknown. Our aim was to assess the contribution of the procoagulant platelet surface, and thus platelet-driven thrombin generation, to the progression of thromboinflammation in the ischemic brain. Approach and Results— We present the characterization of a novel platelet and megakaryocyte-specific TMEM16F (anoctamin 6) knockout mouse. Reflecting Scott syndrome, platelets from the knockout mouse had a significant reduction in procoagulant characteristics that altered thrombin and fibrin generation kinetics. In addition, knockout mice showed significant defects in hemostasis and arterial thrombus formation. However, infarct volumes in a model of ischemic stroke were comparable with wild-type mice. Conclusions— Platelet TMEM16F activity contributes significantly to hemostasis and thrombosis but not cerebral thromboinflammation. These results highlight another key difference between the roles of platelets and coagulation in these processes.

Animal modelling for infectious diseases is critical to understand the biology of the pathogens including viruses and to develop therapeutic strategies against it. Herein, we present the sequence homology and expression data analysis of proteins found in Drosophila melanogaster that are orthologous to human proteins, reported as components of SARS-CoV-2/Human interactome. The dataset enlists sequence homology, query coverage, domain conservation, OrthoMCL and Ensembl Genome Browser support of 326 proteins in D.melanogaster that are potentially orthologous to 417 human proteins reported for their direct physical interactions with 28 proteins encoded by SARS-CoV-2 genome. Expression of these D.melanogaster orthologous genes in 26 anatomical positions are also plotted as heat maps in 27 sets, corresponding to the potential protein interactors for each viral protein. The data could be used to direct experiments and potentially predict their phenotypic and molecular outcome in order to dissect the biological roles and molecular functionality of SARS-CoV-2 proteins in a convenient animal model system like D.melanogaster.

Blood platelets are produced by large bone marrow (BM) precursor cells, megakaryocytes (MKs), which extend cytoplasmic protrusions (proplatelets) into BM sinusoids. The molecular cues that control MK polarization towards sinusoids and limit transendothelial crossing to proplatelets remain unknown. Here, we show that the small GTPases Cdc42 and RhoA act as a regulatory circuit downstream of the MK-specific mechanoreceptor GPIb to coordinate polarized transendothelial platelet biogenesis. Functional deficiency of either GPIb or Cdc42 impairs transendothelial proplatelet formation. In the absence of RhoA, increased Cdc42 activity and MK hyperpolarization triggers GPIb-dependent transmigration of entire MKs into BM sinusoids. These findings position Cdc42 (go-signal) and RhoA (stop-signal) at the centre of a molecular checkpoint downstream of GPIb that controls transendothelial platelet biogenesis. Our results may open new avenues for the treatment of platelet production disorders and help to explain the thrombocytopenia in patients with Bernard–Soulier syndrome, a bleeding disorder caused by defects in GPIb-IX-V., Platelets derive from large precursor cells (megakaryocytes) in the bone marrow. Dütting et al. show that megakaryocyte polarization and platelet biogenesis in the bone-marrow sinusoids are directed by adhesion receptor GPIb signalling and resulting balanced antagonism between RhoA (stop-signal) and Cdc42 (go-signal).

Background: Conventional manual semen analysis is done routinely in most reproductive laboratories. Computer-assisted semen analysis (CASA), on the other hand, is more objective and timesaving counterpart to the conventional method. Different CASA software systems are available, some are fully automated. Thus, it is essential to analyze its performance and compare it with the conventional semen analysis method. Aims and Objectives: Our objective was to analyze the same semen sample by manual method using Makler chamber and using sophisticated CASA software. Our aim was to find the level agreement between the two methods. Materials and Methods: Ethical clearance was taken before the study. All the subjects were given proper instructions on how to collect complete semen sample. n = 60 diagnosed primary infertile male patients referred from the obstetrics and gynecology department. Semen of the same patient was analyzed by manual method and CASA software in accordance with the WHO guidelines. The results obtained were analyzed using Pearson’s correlation and Bland and Altman plot. Results: On statistical analysis, “r” value obtained is 0.99 which is highly statistically significant. CASA and manual methods both agree with each other as proved by Bland and Altman plot. Conclusion: Both methods can be used to screen semen samples in reproductive laboratories. Manual method is easy, feasible, requires expert analysis, and has subjective variability. CASA, on the other hand, is faster newer method with better reproducibility.

SummaryStudies on the effects of estrogen-progestrone levels on the MI of the squamous epithelium of vagina of pregnant rats were made. We observed fluctuations in MI for different periods of gestation as well as variations in cytological criteria which occurred especially on days 7 and 14. The MI shifts and marked alterations of the cytological criteria coincides on the same gestational days.