Confinement in fibrous environments positions and orients mitotic spindles.

Accurate positioning of the mitotic spindle within the rounded cell body is critical to physiological maintenance. Adherent mitotic cells encounter confinement from neighboring cells or the extracellular matrix (ECM), which can cause rotation of mitotic spindles and, consequently, titling of the metaphase plate (MP). To understand the positioning and orientation of mitotic spindles under confinement by fibers (ECM-confinement), we use flexible ECM-mimicking nanofibers that allow natural rounding of the cell body while confining it to differing levels. Rounded mitotic bodies are anchored in place by actin retraction fibers (RFs) originating from adhesion clusters on the ECM-mimicking fibers. We discover the extent of ECM-confinement patterns RFs in 3D: triangular and band-like at low and high confinement, respectively. A stochastic Monte-Carlo simulation of the centrosome (CS), chromosome (CH), membrane interactions, and 3D arrangement of RFs on the mitotic body recovers MP tilting trends observed experimentally. Our mechanistic analysis reveals that the 3D shape of RFs is the primary driver of the MP rotation. Under high ECM-confinement, the fibers can mechanically pinch the cortex, causing the MP to have localized deformations at contact sites with fibers. Interestingly, high ECM-confinement leads to low and high MP tilts, which mechanistically depend upon the extent of cortical deformation, RF patterning, and MP position. We identify that cortical deformation and RFs work in tandem to limit MP tilt, while asymmetric positioning of MP leads to high tilts. Overall, we provide fundamental insights into how mitosis may proceed in fibrous ECM-confining microenvironments in vivo.

CKAP5 stabilizes CENP-E at kinetochores by regulating microtubule-chromosome attachments.

Stabilization of microtubule plus end-directed kinesin CENP-E at the metaphase kinetochores is important for chromosome alignment, but its mechanism remains unclear. Here, we show that CKAP5, a conserved microtubule plus tip protein, regulates CENP-E at kinetochores in human cells. Depletion of CKAP5 impairs CENP-E localization at kinetochores at the metaphase plate and results in increased kinetochore-microtubule stability and attachment errors. Erroneous attachments are also supported by computational modeling. Analysis of CKAP5 knockout cancer cells of multiple tissue origins shows that CKAP5 is preferentially essential in aneuploid, chromosomally unstable cells, and the sensitivity to CKAP5 depletion is correlated to that of CENP-E depletion. CKAP5 depletion leads to reduction in CENP-E-BubR1 interaction and the interaction is rescued by TOG4-TOG5 domain of CKAP5. The same domain can rescue CKAP5 depletion-induced CENP-E removal from the kinetochores. Interestingly, CKAP5 depletion facilitates recruitment of PP1 to the kinetochores and furthermore, a PP1 target site-specific CENP-E phospho-mimicking mutant gets stabilized at kinetochores in the CKAP5-depleted cells. Together, the results support a model in which CKAP5 controls mitotic chromosome attachment errors by stabilizing CENP-E at kinetochores and by regulating stability of the kinetochore-attached microtubules.

Active XY model on a substrate: Density fluctuations and phase ordering.

We explore the generic long-wavelength properties of an active XY model on a substrate, consisting of a collection of nearly phase-ordered active XY spins in contact with a diffusing, conserved species, as a representative system of active spinners with a conservation law. The spins rotate actively in response to the local density fluctuations and local phase differences, on a solid substrate. We investigate this system by Monte Carlo simulations of an agent-based model, which we set up, complemented by the hydrodynamic theory for the system. We demonstrate that this system can phase-synchronize without any hydrodynamic interactions. Our combined numerical and analytical studies show that this model, when stable, displays hitherto unstudied scaling behavior: As a consequence of the interplay between the mobility, active rotation, and number conservation, such a system can be stable over a wide range of the model parameters characterized by a novel correspondence between the phase and density fluctuations. In different regions of the phase space where the phase-ordered system is stable, it displays generalized quasi-long-range order (QLRO): It shows phase ordering which is generically either logarithmically stronger than the conventional QLRO found in its equilibrium limit, together with "miniscule number fluctuations," or logarithmically weaker than QLRO along with "giant number fluctuations," showing a novel one-to-one correspondence between phase ordering and density fluctuations in the ordered states. Intriguingly, these scaling exponents are found to depend explicitly on the model parameters. We further show that in other parameter regimes there are no stable, ordered phases. Instead, two distinct types of disordered states with short-range phase order are found, characterized by the presence or absence of stable clusters of finite sizes. In a surprising connection, the hydrodynamic theory for this model also describes the fluctuations in a Kardar-Parisi-Zhang (KPZ) surface with a conserved species on it, or an active fluid membrane with a finite tension, without momentum conservation and a conserved species living on it. This implies the existence of stable fluctuating surfaces that are only logarithmically smoother or rougher than the Edward-Wilkinson surface at two dimensions (2D) can exist, in contrast to the 2D pure KPZ-like "rough" surfaces.

Mobility-induced order in active XY spins on a substrate.

We elucidate that the nearly phase-ordered active XY spins in contact with a conserved, diffusing species on a substrate can be stable. For wide-ranging model parameters, it has stable uniform phases robust against noises. These are distinguished by generalized quasi-long-range (QLRO) orientational order logarithmically stronger or weaker than the well-known QLRO in equilibrium, together with miniscule (i.e., hyperuniform) or giant number fluctuations, respectively. This illustrates a direct correspondence between the two. The scaling of both phase and density fluctuations in the stable phase-ordered states is nonuniversal: they depend on the nonlinear dynamical couplings. For other parameters, it has no stable uniformly ordered phase. Our model, a theory for active spinners, provides a minimal framework for wide-ranging systems, e.g., active superfluids on substrates, synchronization of oscillators, active carpets of cilia and bacterial flagella, and active membranes.

Comparison of SARS-CoV-2 diagnosis by rapid antigen detection kit with RT-qPCR in a tertiary care setup in North Eastern India.

PURPOSE: Real time reverse transcriptase polymerase chain reaction (RT-qPCR) is still considered a gold standard for the diagnosis of COVID-19. However, due to several limitations, use of RT-qPCR is limited in a resource poor setting like North East India. Rapid antigen detection testing kit has revolutionized the diagnosis and management of COVID-19 in India. However, conflicting reports exist regarding the efficacy of the kits for diagnosis of COVID-19. This study aims to highlight the performance of Standard Q COVID-19® Antigen detection kit (SD Biosensor) compared with RT-qPCR in the setup of North East India. METHODS: Nasopharyngeal and oropharyngeal swab samples were collected from consenting patients attending the flu clinic in the period from 1st July to December 31, 2020. Samples were transferred to Viral Research and Diagnostic Laboratory (VRDL) for RT-qPCR test. Antigen detection from the patient samples were undertaken using Standard Q ® COVID-19 antigen detection kit (SD Biosensor, Republic of Korea). Data were then analyzed for comparison between RT-qPCR and antigen kit results. RESULTS: During the study period, 189 samples were collected, out of which 119 were positive by RT-qPCR. Out of 119 positive samples, calculated sensitivity and specificity of the rapid antigen kit was 63% and 100% respectively. Sensitivity and diagnostic accuracy increases in symptomatic patients as compared to asymptomatic patients. Cohen's Kappa coefficient showed a moderate association (0.6) between the kit and RT-qPCR test. The kit performed optimally at a CT value of ≤32.5 for N gene with a predicted sensitivity of 77.3% and specificity of 93.3%. CONCLUSION: The study shows an overall acceptable sensitivity and specificity of the testing kit, with a better performance in symptomatic patients. The association of the kit result is moderate with the results obtained in RT-qPCR. In this study, the rapid antigen test kit performed optimally at N gene qRT PCR cut off value of ≤32.5.

Mitotic outcomes and errors in fibrous environments.

During mitosis, cells round up and utilize the interphase adhesion sites within the fibrous extracellular matrix (ECM) as guidance cues to orient the mitotic spindles. Here, using suspended ECM-mimicking nanofiber networks, we explore mitotic outcomes and error distribution for various interphase cell shapes. Elongated cells attached to single fibers through two focal adhesion clusters (FACs) at their extremities result in perfect spherical mitotic cell bodies that undergo significant 3-dimensional (3D) displacement while being held by retraction fibers (RFs). Increasing the number of parallel fibers increases FACs and retraction fiber-driven stability, leading to reduced 3D cell body movement, metaphase plate rotations, increased interkinetochore distances, and significantly faster division times. Interestingly, interphase kite shapes on a crosshatch pattern of four fibers undergo mitosis resembling single-fiber outcomes due to rounded bodies being primarily held in position by RFs from two perpendicular suspended fibers. We develop a cortex-astral microtubule analytical model to capture the retraction fiber dependence of the metaphase plate rotations. We observe that reduced orientational stability, on single fibers, results in increased monopolar mitotic defects, while multipolar defects become dominant as the number of adhered fibers increases. We use a stochastic Monte Carlo simulation of centrosome, chromosome, and membrane interactions to explain the relationship between the observed propensity of monopolar and multipolar defects and the geometry of RFs. Overall, we establish that while bipolar mitosis is robust in fibrous environments, the nature of division errors in fibrous microenvironments is governed by interphase cell shapes and adhesion geometries.

Diagnosis of Indigenous Non-Malarial Vector-Borne Infections from Malaria Negative Samples from Community and Rural Hospital Surveillance in Dhalai District, Tripura, North-East India.

The aetiology of non-malaria vector-borne diseases in malaria-endemic, forested, rural, and tribal-dominated areas of Dhalai, Tripura, in north-east India, was studied for the first time in the samples collected from malaria Rapid Diagnostic Kit negative febrile patients by door-to-door visits in the villages and primary health centres. Two hundred and sixty serum samples were tested for the Dengue NS1 antigen and the IgM antibodies of Dengue, Chikungunya, Scrub Typhus (ST), and Japanese Encephalitis (JE) during April 2019-March 2020. Fifteen Dengue, six JE, twelve Chikungunya, nine ST and three Leptospirosis, and mixed infections of three JE + Chikungunya, four Dengue + Chikungunya, three Dengue + JE + Chikungunya, one Dengue + Chikungunya + ST, and one Dengue + ST were found positive by IgM ELISA tests, and four for the Dengue NS1 antigen, all without any travel history. True prevalence values estimated for infections detected by Dengue IgM were 0.134 (95% CI: 0.08-0.2), Chikungunya were 0.084 (95% CI: 0.05-0.13), Scrub were 0.043 (95% CI: 0.01-0.09), and Japanese Encephalitis were 0.045 (95% CI: 0.02-0.09). Dengue and Chikungunya were associated significantly more with a younger age. There was a lack of a defined set of symptoms for any of the Dengue, Chikungunya, JE or ST infections, as indicated by the k-modes cluster analysis. Interestingly, most of these symptoms have an overlapping set with malaria; thereby, it becomes imperative that malaria and these non-malaria vector-borne disease diagnoses are made in a coordinated manner. Findings from this study call for advances in routine diagnostic procedures and the development of a protocol that can accommodate, currently, in practicing the rapid diagnosis of malaria and other vector-borne diseases, which is doable even in the resource-poor settings of rural hospitals and during community fever surveillance.

Capillary Hemangioma of the External Auditory Canal Extending to Middle Ear and Mastoid Cavity: A Rare Case Report with Review of Literature.

Capillary hemangioma  involving external auditory canal, middle ear and mastoid cavity is a very rare entity. Due to infrequent incidence but multiple overlapping clinical and radiological features amongst some common middle ear pathologies this benign vascular tumour often can be misdiagnosed. Histopathological report helps us to get definitive diagnosis. 40 years old female presented with diminished hearing and mass in right ear cavity for last 3 years. The otoscopic examination of right ear showed a pinkish polypoidal mass at the external auditory canal and tympanic membrane could not be visualized. Patient had a history of cortical mastoidectomy 2 years back for the similar problem. HRCT temporal bone revealed a single cavity with soft tissue density content in right mastoid cavity which was extending into right middle ear and external ear. Contrast Enhanced MRI showed a long polypoidal heterogeneously but strong enhancing T2 and STIR hyperintense lesion within mastoid antrum extending into middle ear cavity and external auditory canal forming an aural polyp. Excision of the mass was done by canal wall down mastoidectomy and also type IIIc tympanoplasty performed in same sitting. There is no recurrence noted after 6 months of follow up and hearing improvement noticed. Capillary haemangioma of mastoid antrum extending to middle ear and external auditory canal requires surgical excision as preferred treatment modality, pre-operative imaging guide us to choose the preferred surgical approaches.

Studying the progress of COVID-19 outbreak in India using SIRD model.

We explore a standard epidemiological model, known as the SIRD model, to study the COVID-19 infection in India, and a few other countries around the world. We use (a) the stable cumulative infection of various countries and (b) the number of infection versus the tests carried out to evaluate the model. The time-dependent infection rate is set in the model to obtain the best fit with the available data. The model is simulated aiming to project the probable features of the infection in India, various Indian states, and other countries. India imposed an early lockdown to contain the infection that can be treated by its healthcare system. We find that with the current infection rate and containment measures, the total active infection in India would be maximum at the end of June or beginning of July 2020. With proper containment measures in the infected zones and social distancing, the infection is expected to fall considerably from August. If the containment measures are relaxed before the arrival of the peak infection, more people from the susceptible population will fall sick as the infection is expected to see a threefold rise at the peak. If the relaxation is given a month after the peak infection, a second peak with a moderate infection will follow. However, a gradual relaxation of the lockdown started well ahead of the peak infection, leads to a nearly twofold increase of the peak infection with no second peak. The model is further extended to incorporate the infection arising from the population showing no symptoms. The preliminary finding suggests that random testing needs to be carried out within the asymptomatic population to contain the spread of the disease. Our model provides a semi-quantitative overview of the progression of COVID-19 in India, with model projections reasonably replicating the current progress. The projection of the model is highly sensitive to the choice of the parameters and the available data.

COVID-19: District level vulnerability assessment in India.

OBJECTIVES: COVID-19 Pandemic has brought a threatening challenge to the world and as well as for Indian society and economy. In India, it has become a public health disaster and its' intensity increasing continuously. For the disaster risk reduction, and capacity building against the COVID-19 pandemic understanding of the relationship between socio-environmental conditions with the pandemic is very necessary. The objective of the present work is to construct a socio-environmental vulnerability index of the potential risk of community spread of COVID-19 using socio-economic and environmental variables. METHODOLOGY: In this, cross-sectional study principal component analyses have been used to drive SoEVI. 4 uncorrelated sub-index has been extracted from 16 sub-indicators which reflects 59% of the variance. Aggregation of 4 Sub-Index has been done to obtain the final vulnerability Index. RESULTS: Results show that there is spatial variability in vulnerability based on environmental and socio-economic conditions. Districts of north and central India found more vulnerable then south India. Statistical significance has been tested using regression analysis, positive relation has been found between vulnerability index and confirmed and active cases. CONCLUSION: The vulnerability index has highlighted environmentaly and socioeconomicallybackward districts. These areas will suffer more critical problems against COVID-19 pandemic for their socio-environmental problem.

SEIRD model to study the asymptomatic growth during COVID-19 pandemic in India.

According to the current perception, symptomatic, presymptomatic and asymptomatic infectious persons can infect the healthy population susceptible to the SARS-CoV-2. More importantly, various reports indicate that the number of asymptomatic cases can be several-fold higher than the reported symptomatic cases. In this article, we take the reported cases in India and various states within the country till September 1, as the specimen to understand the progression of the COVID-19. Employing a modified SEIRD model, we predict the spread of COVID-19 by the symptomatic as well as asymptomatic infectious population. Considering reported infection primarily due to symptomatic, we compare the model predicted results with the available data to estimate the dynamics of the asymptomatically infected population. Our data indicate that in the absence of the asymptomatic infectious population, the number of symptomatic cases would have been much less. Therefore, the current progress of the symptomatic infection can be reduced by quarantining the asymptomatically infectious population via extensive or random testing. This study is motivated strictly toward academic pursuit; this theoretical investigation is not meant for influencing policy decisions or public health practices.

COVID-19 second wave: District level study of concentration of confirmed cases and fatality in India.

The paper aims to reveal the spatial pattern of the concentration of COVID-19 confirmed cases and the spread of the pandemic from the Case Fatality Ratio. The study has been accomplished with district-level data. The analysis of the spatial pattern decoding has been done considering the Global and Local Moran's I statistics comprising the linear trend of spatial autocorrelation for the whole India. The timeframe has been divided considering the surge of the second wave in March, 2021 and the peak of the wave in May 2021. The spatial clustering technique presents both the concentration of confirmed cases using Location Quotient analysis and the pattern of spread of the infection-related fatality throughout the country. The high Location Quotient of the confirmed cases strongly clustered around the Mumbai-Puna region, Kerala-Karnataka region, Garhwal Himachal, NCT of Delhi and Ladakh-Kashmir-Himachal Pradesh region during the period of the study. In May, the concentration has randomly clustered around the middle part of India. The Case Fatality Ratio was high in Maharashtra, Madhya Pradesh, Punjab and Haryana at the surge of the second wave. During the peak (May), two significant clusters of high Case Fatality Ratio are observed in and around the Mumbai urban (Maharashtra) and NCT of Delhi (including Punjab-Haryana).

Evidence of positive regulation of mycobacteriophage D29 early gene expression obtained from an investigation using a temperature-sensitive mutant of the phage.

Mycobacteriophages are phages that infect and kill Mycobacteria, several of which, Mycobacterium tuberculosis (Mtb), for example, cause the disease tuberculosis. Although genomes of many such phages have been sequenced, we have very little insight into how they express their genes in a controlled manner. To address this issue, we have raised a temperature-sensitive (ts) mutant of phage D29 that can grow at 37°C but not at 42°C and used it to perform differential gene expression and proteome analysis studies. Our analysis results indicate that expression of genes located in the right arm, considered to be early expressed, was lowered as the temperature was shifted from 37°C to 42°C. In contrast, expression of those on the left, the late genes were only marginally affected. Thus, we conclude that transcription of genes from the two arms takes place independently of each other and that a specific factor must be controlling the expression of the right arm genes. We also observe that within the right arm itself; there exists a mechanism to ensure high-level synthesis of Gp48, a thymidylate synthase X. Enhanced presence of this protein in infected cells results in delayed lysis and higher phage yields.

Mechanics of Multicentrosomal Clustering in Bipolar Mitotic Spindles.

To segregate chromosomes in mitosis, cells assemble a mitotic spindle, a molecular machine with centrosomes at two opposing cell poles and chromosomes at the equator. Microtubules and molecular motors connect the poles to kinetochores, specialized protein assemblies on the centromere regions of the chromosomes. Bipolarity of the spindle is crucial for the proper cell division, and two centrosomes in animal cells naturally become two spindle poles. Cancer cells are often multicentrosomal, yet they are able to assemble bipolar spindles by clustering centrosomes into two spindle poles. Mechanisms of this clustering are debated. In this study, we computationally screen effective forces between 1) centrosomes, 2) centrosomes and kinetochores, 3) centrosomes and chromosome arms, and 4) centrosomes and cell cortex to understand mechanics that determines three-dimensional spindle architecture. To do this, we use the stochastic Monte Carlo search for stable mechanical equilibria in the effective energy landscape of the spindle. We find that the following conditions have to be met to robustly assemble the bipolar spindle in a multicentrosomal cell: 1) the strengths of centrosomes' attraction to each other and to the cell cortex have to be proportional to each other and 2) the strengths of centrosomes' attraction to kinetochores and repulsion from the chromosome arms have to be proportional to each other. We also find that three other spindle configurations emerge if these conditions are not met: 1) collapsed, 2) monopolar, and 3) multipolar spindles, and the computational screen reveals mechanical conditions for these abnormal spindles.

Mycobacterium tuberculosis thymidylate synthase (ThyX) is a target for plumbagin, a natural product with antimycobacterial activity.

Plumbagin derived from the plant Plumbago indica, known as Chitrak in India, is an example of a medicinal compound used traditionally to cure a variety of ailments. Previous reports have indicated that it can inhibit the growth of Mycobacterium tuberculosis (Mtb), the causative agent of the deadly disease TB. In this investigation, we provide an insight into its mode of action. We show here that a significant mycobacterial target that is inhibited by plumbagin is the enzyme ThyX, a form of thymidylate synthase, that is responsible for the synthesis of dTMP from dUMP in various bacterial pathogens, including Mtb. Using a purified preparation of the recombinant version of Mtb ThyX, we demonstrate that plumbagin, a 2,4 napthoquinone, but not lawsone, a structurally related medicinal compound, inhibits its activity in vitro. We also show that the intracellular [dTTP]/[dATP] ratio in Mycobacterium smegmatis (Msm) cells decrease upon treatment with plumbagin, and this, in turn, leads to cell death. Such a conclusion is supported by the observation that over-expression of thyx in the plumbagin treated Msm cells leads to the restoration of viability. The results of our investigation indicate that plumbagin kills mycobacterial cells primarily by targeting ThyX, a vital enzyme required for their survival.

Search and Capture Efficiency of Dynamic Microtubules for Centrosome Relocation during IS Formation.

Upon contact with antigen-presenting cells, cytotoxic T lymphocytes (T cells) establish a highly organized contact zone denoted as the immunological synapse (IS). The formation of the IS implies relocation of the microtubule organizing center (MTOC) toward the contact zone, which necessitates a proper connection between the MTOC and the IS via dynamic microtubules (MTs). The efficiency of the MTs finding the IS within the relevant timescale is, however, still illusive. We investigate how MTs search the three-dimensional constrained cellular volume for the IS and bind upon encounter to dynein anchored at the IS cortex. The search efficiency is estimated by calculating the time required for the MTs to reach the dynein-enriched region of the IS. In this study, we develop simple mathematical and numerical models incorporating relevant components of a cell and propose an optimal search strategy. Using the mathematical model, we have quantified the average search time for a wide range of model parameters and proposed an optimized set of values leading to the minimal capture time. Our results show that search times are minimal when the IS formed at the nearest or at the farthest sites on the cell surface with respect to the perinuclear MTOC. The search time increases monotonically away from these two specific sites and is maximal at an intermediate position near the equator of the cell. We observed that search time strongly depends on the number of searching MTs and distance of the MTOC from the nuclear surface.