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There is an increased trend of e-cigarette but the toxic effects of e-cigarette metabolites are not widely studied especially in liver disease. Hence, we aimed to evaluate the prevalence and patterns of recent e-cigarette use in a nationally representative sample of US adults and adolescents and its association amongst respondents with liver disease.We conducted a retrospective cross-sectional study using National Health and Nutrition Examination Survey (NHANES) database from 2015 to 2018. The self-reported NHANES questionnaire was used to assess liver disease (MCQ160L, MCQ170L and MCQ 510 (a-e)), e-cigarette use (SMQ900) and traditional smoking status (SMQ020 or SMQ040). We conducted univariate analysis and multivariable logistic regression models to predict the association of e-cigarette use, traditional smoking and dual smoking amongst the population with liver disease.Out of total 178,300 respondents, 7,756 (4.35%) were e-cigarette users, 48,625 (27.27%) traditional smoking, 23,444 (13.15%) dual smoking and 98,475 (55.23%) non-smokers. Females had a higher frequency of e-cigarette use (49.3%) compared to dual (43%) and traditional smoking (40.8%) (P0.0001). Respondents with a past history of any liver disease have lower frequency of e-cigarette use compared to dual and traditional smoking, respectively (2.4% vs. 6.4% vs. 7.2%; P0.0001). In multivariate logistic regression models, we found that e-cigarette users (odds ratio (OR): 1.06; 95% confidence interval (CI): 1.05 - 1.06; P0.0001) and dual smoking (OR: 1.50; 95% CI: 1.50 - 1.51; P0.0001) were associated with higher odds of having history of liver disease compared to non-smokers.Our study found that despite the low frequency of e-cigarette use in respondents with liver disease, there was higher odds of e-cigarette use amongst patients with liver disease. This warrants the need for more future prospective studies to evaluate the long-term effects and precise mechanisms of e-cigarette toxicants on the liver.

Myxomas, being the most common primary benign tumor among all cardiac tumors, occur rarely with a very low incidence rate. Myxomas can cause various clinical manifestations or can be diagnosed incidentally. Some patients with cardiac myxomas are completely asymptomatic. Cardiac myxomas cause life-threatening complications, thus early diagnosis is imperative. We are reporting a case of atrial myxoma in a 38-year-old female without any significant past medical history, who came to our clinic for cardiology evaluation prior to plastic surgery. The elliptical mobile mass, located in the left atrium with its attachment to the interatrial septum, was diagnosed by transthoracic echocardiography. The patient was referred for surgery and a minimally invasive surgical approach was chosen. A histological report confirmed the diagnosis of myxoma. The patient recovered well. Three years of follow-up did not reveal any signs of recurrence of the tumor. We are also analyzing 20 previously published cases of asymptomatic myxomas and myxomas treated with a minimally invasive surgical approach, reported in the PubMed database for the last 20 years (2001-2021) in the adult patient population (age 19 and over). The aim of this study is to highlight the asymptomatic presentation of cardiac myxomas and to underline the advantages of a minimally invasive surgical approach. In summary, asymptomatic cardiac myxomas are rare incidental findings. Attention to subtle symptoms during a physical exam and scrupulous history-taking can provide a clue to this diagnosis. Early diagnosis of cardiac myxomas is crucial to prevent life-threatening complications. Minimally invasive surgery is a promising alternative approach to standard open-heart surgery for treating cardiac myxomas, providing faster recovery and higher patient satisfaction with surgical care.

There is an established literature on the symptoms and complications of COVID‐19 but the after‐effects of COVID‐19 are not well understood with few studies reporting persistent symptoms and quality of life. We aim to evaluate the pooled prevalence of poor quality of life in post‐acute COVID‐19 syndrome (PCS) and conducted meta‐regression to evaluate the effects of persistent symptoms and intensive care unit (ICU) admission on the poor quality of life. We extracted data from observational studies describing persistent symptoms and quality of life in post‐COVID‐19 patients from March 10, 2020, to March 10, 2021, following PRISMA guidelines with a consensus of two independent reviewers. We calculated the pooled prevalence with 95% confidence interval (CI) and created forest plots using random‐effects models. A total of 12 studies with 4828 PCS patients were included. We found that amongst PCS patients, the pooled prevalence of poor quality of life (EQ‐VAS) was (59%; 95% CI: 42%–75%). Based on individual factors in the EQ‐5D‐5L questionnaire, the prevalence of mobility was (36, 10–67), personal care (8, 1–21), usual quality (28, 2–65), pain/discomfort (42, 28–55), and anxiety/depression (38, 19–58). The prevalence of persistent symptoms was fatigue (64, 54–73), dyspnea (39.5, 20–60), anosmia (20, 15–24), arthralgia (24.3, 14–36), headache (21, 3–47), sleep disturbances (47, 7–89), and mental health (14.5, 4–29). Meta‐regression analysis showed the poor quality of life was significantly higher among post‐COVID‐19 patients with ICU admission (p = 0.004) and fatigue (p = 0.0015). Our study concludes that PCS is associated with poor quality of life, persistent symptoms including fatigue, dyspnea, anosmia, sleep disturbances, and worse mental health. This suggests that we need more research on PCS patients to understand the risk factors causing it and eventually leading to poor quality of life.

RATIONAL: Coronaviruses are RNA viruses that primarily affect the respiratory system. Patients with Coronavirus Disease-19 (COVID-19) pneumonia and respiratory distress often require hospitalization, ICU admission, intubation and/or non-invasive ventilation, and circulatory support. Some experts suggest that the pathophysiology of traditional ARDS and that of the lung disease associated with COVID-19 are different. It is proposed that the severe hypoxemia in COVID-19 patients is the result of hypoxia-induced vasoconstriction and a large burden of microthrombi which result in intrapulmonary shunting. Therefore, COVID-related ARDS-like patterns of disease may not respond to World Health Organization (WHO) recommendations for early intubation and ARDS Network (ARDSNet) recommendations for FiO2-titrated Positive End-Expiratory Pressure (PEEP) administration and ventilator support. Given the distinct pathophysiology of COVID-19-related ARDS, higher PEEP may indicate mechanical over-inflation of the alveoli resulting in volume trauma in addition to compromising preload and decreasing cardiac output, worsening the pulmonary hypoperfusion. The aim of this study was to examine the rate of death associated with initial and subsequent (48-hour) PEEP settings of 10cmH2O and higher in COVID-19 patients. We hypothesized that higher PEEP settings may lead to increased mortality. METHODS: We conducted a retrospective chart review of patients who presented or were transferred to our facility with COVID-19 and were subsequently intubated from March 1st until April 30th, 2020. Charts were reviewed for initial and 48-hour PEEP settings. Mortality, SARS CoV2 results, clinical data and demographics were also recorded. RESULTS: A total of 74 patients were included in the review. Of these, 53 patients had initial PEEP setting of >10cmH2O and 46 patients had PEEP settings >10cmH2O 48 hours after intubation. Thirty-seven patients had PEEP settings >10cmH2O both initially and 48 hours later. Thirty-seven patients who had an initial PEEP setting of >10 cmH220 died, (70%). Forty patients who had PEEP settings >10cmH2O at 48hrs died (87%). Twenty-seven patients who had both a PEEP setting >10 cmH2O initially and at 48 hours died (73%). CONCLUSIONS: There appears to be an association in COVID-19 patients between high mortality rates and high PEEP settings. The marked association is strong enough, in our view, to suggest that conventional ventilator settings recommended by ARDSNet may not apply to patients experiencing COVID-19-associated ARDS and in fact, may be leading to worse patient outcomes.

Introduction: Literature reported, 9-17% transient ischemic attack (TIA) patients have a vascular event within 90 days but there is limited data on long term risk involvement of vascular events following TIA. Aim: To identify prevalence and association of cardiovascular diseases (CVD) and cerebrovascular disorders (CeVD) amongst early and late TIA. Methods: We performed a retrospective cross-sectional analysis of Nationwide Inpatient Sample (2016-2017) in adults hospitalizations. Early TIA (primary diagnosis), late TIA (secondary diagnosis-within a year) and vascular events [CVD (AFib, IHD, acute MI angina) and CeVD (AIS, ICeH, SAH)] amongst TIA were identified using ICD 10 CM codes. Prevalence of vascular events were compared amongst patients with TIA and without TIA. Weighted analysis to account for sampling strategy using mix-effect multivariable survey logistic regression was performed to evaluate odds of having vascular events amongst TIA in comparison to non-TIA. Results: Amongst 58,259,589 US hospitalizations, 0.38% and 5.92% patients had early and late TIA, respectively. Patients with late TIA had higher prevalence of acute MI (4.9 vs 0.5 vs 3.4%), IHD (44 vs 28.6 vs 20.6%), angina (0.3 vs 0.2 vs 0.2%), AFib (22 vs 15.3 vs 10.9%), AIS (5.3 vs 0.6 vs 2%), SAH (0.2 vs 0.03 vs 0.1%) and ICeH (0.8 vs 0.04 vs 0.4%) compared to early TIA and no-TIA, respectively. (p(Table 1) Conclusion: We found a higher prevalence of late TIA amongst US hospitalizations. Additionally, late TIA patients had a higher risk of vascular events like Afib, IHD, and stroke. Hence, a thorough clinical investigation and long term followup of TIA patients may mitigate the risk of future vascular events and associated health care burden.

Introduction There is limited literature on coronavirus disease 2019 (COVID -19) complications such as thromboembolism, cardiac complications etc. as possible trigger for stroke. Hence, we aim to evaluate the prevalence and outcomes of COVID-19 related cardiovascular complications and secondary infection and their possibility as potential triggers for the stroke. Methods Data from observational studies describing the complications [acute cardiac injury (ACI), cardiac arrhythmias (CA), disseminated intravascular coagulation (DIC), septic shock, secondary infection] and outcomes of COVID‐19 hospitalized patients from December 1, 2019 to June 30, 2020, were extracted following PRISMA guidelines. Adverse outcomes defined as intensive care units, oxygen saturation less than 90%, invasive mechanical ventilation, severe disease, and in‐hospital mortality. The odds ratio and 95% confidence interval were obtained, and forest plots were created using random‐effects models. A short review of these complications as triggers of stroke was conducted. Results 16 studies with 3480 confirmed COVID-19 patients, prevalence of ACI [38%vs5.9%], CA [26%vs5.3%], DIC [4%vs0.74%], septic shock [18%vs0.36%], and infection [30%vs12.5%] was higher among patients with poor outcomes. In meta-analysis, ACI [aOR:9.93(95%CI:3.95–25.00], CA [7.52(3.29–17.18)], DIC [7.36(1.24–43.73)], septic shock [30.12(7.56–120.10)], and infection [10.41(4.47–24.27)] had higher odds of adverse outcomes. Patients hospitalized with acute ischemic stroke and intracerebral hemorrhage, had complications like pulmonary embolism, venous thromboembolism, DIC, etc. and had poor outcomes Conclusion The complications like acute cardiac injury, cardiac arrhythmias, DIC, septic shock, and secondary infection had poor outcomes. Patients with stroke were having history of these complications. Long term monitoring is required in such patients to prevent stroke and mitigate adverse outcomes.

Highlights • In this meta-analysis of 24 studies with 12882 confirmed COVID-19 patients, we assessed the association of comorbid liver disease, acute liver injury, and elevated liver enzymes with outcomes in COVID-19 hospitalized patients. • Overall prevalence of pre-existing chronic liver disease and COVID-19-associated acute liver injury were 2.6% and 26.5%, respectively and elevated AST and ALT were 41.1% and 29.1%, respectively. • In our study, COVID-19-associated acute liver injury was having 1.7 folds higher risk of poor outcomes. • Elevated AST and ALT were also independently associated with higher odds of poor outcomes. • These findings may help in early triage, close monitoring of the occurrence of liver injury, and careful use of drugs which can cause liver toxicity in COVID-19 patients., Introduction and Objectives The coronavirus disease 2019 (COVID-19) pandemic has been a challenge globally. In severe acute respiratory syndrome (SARS) epidemic 60% of patients had hepatic injury, due to phylogenetic similarities of the viruses it is assumed that COVID-19 is associated with acute liver injury. In this meta-analysis, we aim to study the occurrence and association of liver injury, comorbid liver disease and elevated liver enzymes in COVID-19 confirmed hospitalizations with outcomes. Materials and Methods Data from observational studies describing comorbid chronic liver disease, acute liver injury, elevated aspartate aminotransferase (AST), alanine aminotransferase (ALT) levels and outcomes of COVID-19 hospitalized patients from December 1, 2019, to June 30, 2020 was extracted following PRISMA guidelines. Adverse outcomes were defined as admission to intensive care unit (ICU), oxygen saturation

Cell motility depends on tight coordination between the microtubule (MT) and actin cytoskeletons, but the mechanisms underlying this MT–actin cross talk have remained poorly understood. Here, we show that the tumor suppressor protein adenomatous polyposis coli (APC), which is a known MT-associated protein, directly nucleates actin assembly to promote directed cell migration. By changing only two residues in APC, we generated a separation-of-function mutant, APC (m4), that abolishes actin nucleation activity without affecting MT interactions. Expression of full-length APC carrying the m4 mutation (APC (m4)) rescued cellular defects in MT organization, MT dynamics, and mitochondrial distribution caused by depletion of endogenous APC but failed to restore cell migration. Wild-type APC and APC (m4) localized to focal adhesions (FAs), and APC (m4) was defective in promoting actin assembly at FAs to facilitate MT-induced FA turnover. These results provide the first direct evidence for APC-mediated actin assembly in vivo and establish a role for APC in coordinating MTs and actin at FAs to direct cell migration.

Lattice location of B in TiO2 is tuned to determine its effect on the photocatalytic activity of Co-B codoped TiO2. Sol–gel method was used to synthesize the samples. The concentrations of Co and B were first optimized by maximizing the photocatalytic activity for the monodoped (Co or B)-TiO2. In addition to the DFT calculations for discovering new energetic levels introduced in TiO2 by codoping, various characterization techniques were used to determine the dopant lattice sites in TiO2 and interactions between them; and also determining their consequences on electronic, morphological, structural, and optical properties. At low concentration of B-doping (1 at.%), B occupies the interstitial site (Bint), but as the concentration increases (2 at.% and 3 at.%) B also occupies substitutional O position (Bsub) in addition to Bint to form TiO2 containing Bint+sub. Both these B-doped TiO2 showed improved photocatalytic activity attributed to effective charge separation obtained for TiO2–Bint due to the formation of shallow energy level while higher visible light absorption is achieved with TiO2–Bint+sub owing to the presence of two deep energy levels in the band gap as confirmed by DFT calculations. In the case of Co doping, the band gap of TiO2 is reduced but the recombination rates are always high and are caused by the formation of Co states in the band gap. For Co monodoped TiO2, the photocatalytic activity is low for all the concentrations considered, except for very low concentration of Co (0.1 at.%). Two opposite effects were observed when small amount of Co (0.1 at.%) was codoped with Bint or Bint+sub. In particular, the photocatalytic degradation rate of organic aqueous pollutants (p-nitrophenol and rhodamine B dye) reduces for TiO2–Co–Bint whereas it is enhanced remarkably for TiO2–Co–Bint+sub as compared to (Co or B) monodoped (∼2.1 times) and undoped (∼7.8 times) TiO2. Higher photocatalytic activity observed in Co-doped TiO2–Bint+sub is discussed in terms of the interactions of Co with B at two different lattice positions in TiO2 and the synergistic effect created by higher visible light absorption and the improved charge separation.

Summary EB1 was discovered 25 years ago as a binding partner of the tumor suppressor adenomatous polyposis coli (APC) [ 1 ]; however, the significance of EB1-APC interactions has remained poorly understood. EB1 functions at the center of a network of microtubule end-tracking proteins (+TIPs) [ 2 , 3 , 4 , 5 ], and APC binding to EB1 promotes EB1 association with microtubule ends and microtubule stabilization [ 6 , 7 ]. Whether EB1 interactions govern functions of APC beyond microtubule regulation has not been explored. The C-terminal basic domain of APC (APC-B) directly nucleates actin assembly, and this activity is required in vivo for directed cell migration and for maintaining normal levels of F-actin [ 8 , 9 , 10 ]. Here, we show that EB1 binds APC-B and inhibits its actin nucleation function by blocking actin monomer recruitment. Consistent with these biochemical observations, knocking down EB1 increases F-actin levels in cells, and this can be rescued by disrupting APC-mediated actin nucleation. Conversely, overexpressing EB1 decreases F-actin levels and impairs directed cell migration without altering microtubule organization and independent of its direct binding interactions with microtubules. Overall, our results define a new function for EB1 in negatively regulating APC-mediated actin assembly. Combining these findings with other recent studies showing that APC interactions regulate EB1-dependent effects on microtubule dynamics [ 7 ], we propose that EB1-APC interactions govern bidirectional cytoskeletal crosstalk by coordinating microtubule and actin dynamics.

Cu–N-codoped TiO 2 photocatalyst is synthesized by sol–gel method to obtain enhanced optical absorption in the visible region. Optimum concentrations of Cu and N were obtained by maximizing the photocatalytic activity for the monodoped (Cu or N) TiO 2 . These optimized concentrations were used for synthesizing (Cu, N)–codoped TiO 2 . The photocatalysts were characterized using XRD, micro-Raman, SEM, XPS, BET surface area analyzer, UV–vis diffuse reflectance spectroscopy and photoluminescence. XPS study suggests the incorporation of Cu 2+ into TiO 2 lattice, which assists N to substitutionally replace oxygen in codoped TiO 2 , while maintaining the anatase phase even after doping. N-doping creates minor variation in the energy band gap of TiO 2 reducing it upto 3.0 eV, while Cu doping was able to narrow the band gap to 2.2 eV mainly due to the localized levels of Cu-3d states and shifting of Ti-3d states to lower energy (due to oxygen vacancies) in the band-gap as deduced from XPS data and confirmed by the DFT calculation. In (Cu, N)–codoped TiO 2 , visible light absorption is higher than the other TiO 2 samples, a feature that is mainly attributed to the formation of an isolated intermediate band (IB) occurring due to the strong hybridization between Cu 3d and N 2p orbitals. This IB contributes to visible light absorption by two step optical transition with the first transition from valence band (VB) to IB and the second from IB to conduction band (CB). The dopant species may also act to reduce the charge carrier recombination by acting as the trapping sites for photogenerated charges. (Cu, N)–codoped TiO 2 was able to degrade Methylene Blue dye and p -Nitrophenol solution under light irradiation with significantly better rate in comparison to monodoped and undoped TiO 2 . High photocatalytic activity is attributed to the presence of IB in the energy band gap of TiO 2 , which creates the synergic effect by higher visible light absorption and lower recombination of photogenerated charges.

Introduction Few studies have addressed the mid to long term impact of Laparoscopic Sleeve Gastrectomy (LSG) on weight loss and obesity associated co-morbidities, particularly in Indian population. The aim of this study is to assess the efficacy of LSG in morbid obesity over 3–7 years follow up. Materials and methods Data of all patients who underwent LSG between January 2008 and March 2015 and completed their at least 1 year follow up till March 2016 was retrospectively reviewed using a prospectively collected database. Results 424 patients undergoing primary LSG were included. The mean age (±2SD) was 39.8 ± 22.5 years and the mean Body Mass Index (BMI) (±2SD) was 46.67 ± 15.8 kg/m 2 . 124 patients (29.2%) were super-obese (BMI >50 kg/m 2 ). The percentage follow-up at 1 year, 3 years, 5 years and 7 years was 78.3%, 66.7%, 42.3% and 38.4% respectively. The mean percentage Excess weight Loss (%EWL) (±2SD) at 1year, 3years, 5years, and 7years was 71.8 (±50.5%), 64.95% (±41.8%), 61.7% (±46.2%) and 57.15% (±50.2%) respectively. The preoperative BMI significantly correlated with %EWL at 5 year (r 2 = 0.107, p = 0.018). The overall complication rate was 5.8%. Early complications included staple line leak (1.2%), bleeding (1.2%), deep venous thrombosis (0.4%) and 30-day mortality (0.21%). Late complications included stricture formation (0.21%) and new onset Gastro-esophageal Reflux Disease (GERD) (2.8%).At 5 years, 83.3% of diabetic patients showed remission while rest showed improvement in glycemic control with decrease in dosage. 69.3% patients showed improvement in hypertension, 100% patients showed improvement in Obstructive Sleep Apnea Syndrome, 75% patients showed improvement in hypothyroidism after surgery. GERD resolved in 62.8% patients while worsened in 11.4% patients. Conclusions LSG has durable weight loss at 5 year with %EWL of 61% and significant resolution of obesity associated co-morbidities.

TiO 2 , N-doped TiO 2 , V-doped TiO 2 , and V–N-codoped TiO 2 thin films have been prepared using RF-magnetron sputtering and their photocatalytic activities have been investigated. The codoping strategy was adopted to decrease both the band gap of TiO 2 and the recombination rate of the photo-generated electron–hole pairs. Low concentration doping with single element (V or N) preserves the anatase dominated phase in TiO 2 film while codoping with V and N produces a mixed phase of nearly equal amount of rutile and anatase as inferred from the XRD and Raman spectroscopy studies. XPS studies reveal that, for N-doped TiO 2 elemental N resides at interstitial lattice positions but codoping with V permits N to reside in both substitutional and interstitial sites in TiO 2 lattice. UV–vis studies indicate that the band gap of TiO 2 (3.2 eV) reduced to 3.0 eV, 2.8 eV and 2.5 eV, by N-doping, V-doping and V–N codoping, respectively. The photocatalytic activity of pure, N-doped, V-doped, and V–N codoped TiO 2 thin films were tested by examining the degradation of methylene blue, chlorophenol and nitrophenol as a function of time. It was observed that the codoped TiO 2 gave the highest photocatalytic activity in comparison to the mono-doped and undoped TiO 2 because of high visible light absorption and possible reduction in the recombination of photo-generated charges. Density of states calculated using density functional theory (DFT) showed that the narrowing of band-gap for the codoped TiO 2 is obtained by the formation of isolated energy levels of V 3d and N 2p states below the conduction band and above the valence band of pure TiO 2 , respectively. While for the mono-doped TiO 2 the narrowing of the band gap is only contributed by impurity levels formed near any one of the band edges. It is concluded that for the codoped TiO 2 , high visible light absorption is caused by the formation of impurity energy states near both the band edges which also act as the trapping sites for both the photo-generated charges to reduce the recombination process.

Background: The aims and objectives were to study the progression from tuberculosis to multi drug resistance-TB in revised national tuberculosis control programme: perspectives from health system care givers.Methods: The study was carried out in TB Sanatorium ITKI, Sadar Hospital Ranchi and RIMS Ranchi. The interview of various health personnel including SAHIYAs was taken using a semi-structured questionnaire based on programmatic management of multi drug resistant tuberculosis guidelines -2016.Results: Among Doctors knowledge level was good compared to other health personnel which had mean value 7.33 (±2.79), laboratory technician 3.45 (±2.64), STS 4.67 (±1.59), Sahiya 2.1 (±0.73). Regarding capacity enhancement level all health personnel needed refresher trainings in which doctors got 4.67 (±1.58), laboratory technician 3.45±2.64, STS 1.72±0.34, and Sahiya 0.5±0.52. Specially sahiya needs training regarding MDR-TB because they are the connecting link between health system and community. Regarding execution level, Doctors got 1.86 (±0.74), laboratory technician 1.64 (±0.56), STS 1.64 (±0.56) and Sahiya (ASHA) 2.2 (±0.44). Sahiya were better than other health personnel at execution level.Conclusions: Advocacy, communication, and social mobilization are important aspects of TB control, Policy makers and administrators should be sensitized for need of adequate and sustained funding for TB control to ensure quality capacity building. They need to provide continuous and quality training of staff at different levels and retention of trained staff and periodic reviews to identify gaps and take corrective steps.

SummaryFilopodia are slender cellular protrusions that dynamically extend and retract to facilitate directional cell migration, pathogen sensing, and cell-cell adhesion [1–4]. Each filopodium contains a rigid and organized bundle of parallel actin filaments, which are elongated at filopodial tips by formins and Ena/VASP proteins [5–10]. However, relatively little is known about how the actin filaments in the filopodial shaft are spatially organized to form a bundle with appropriate dimensions and mechanical properties. Here, we report that the mammalian formin Daam1 (Disheveled-associated activator of morphogenesis 1) is a potent actin-bundling protein and localizes all along the filopodial shaft, which differs from other formins that localize specifically to the tips. Silencing of Daam1 led to severe defects in filopodial number, integrity, and architecture, similar to silencing of the bundling protein fascin. This led us to investigate the potential relationship between Daam1 and fascin. Fascin and Daam1 coimmunoprecipitated from cell extracts, and silencing of fascin led to a striking loss of Daam1 localization to filopodial shafts, but not tips. Furthermore, purified fascin bound directly to Daam1, and multicolor single-molecule TIRF imaging revealed that fascin recruited Daam1 to and stabilized Daam1 on actin bundles in vitro. Our results reveal an unanticipated and direct collaboration between Daam1 and fascin in bundling actin, which is required for proper filopodial formation.

See How They Grow Controlled assembly and disassembly of the actin cytoskeleton is essential for processes such as cell motility, cytokinesis, and tumor metastasis. The formation of new actin filaments appears to involve the protein formin paired with another actin assembly-promoting factor. Breitsprecher et al. (p. 1164 ) used triplecolor single-molecule fluorescence microscopy to visualize actin assembly promoted by the formin, mDia1, and the tumor-suppressor, adenomatous polyposis coli (APC). The two assembly factors interacted directly to initiate filament assembly, after which mDia1 moved with the growing barbed ends while APC remained at the site of nucleation.

Recently, we found that divalent calcium has no detectable effect on the assembly of Mycobacterium tuberculosis FtsZ (MtbFtsZ), whereas it strongly promoted the assembly of Escherichia coli FtsZ (EcFtsZ). While looking for potential calcium binding residues in EcFtsZ, we found a mutation (E93R) that strongly promoted the assembly of EcFtsZ. The mutation increased the stability and bundling of the FtsZ protofilaments and produced a dominating effect on the assembly of the wild type FtsZ (WT-FtsZ). Although E93R-FtsZ was found to bind to GTP similarly to the WT-FtsZ, it displayed lower GTPase activity than the WT-FtsZ. E93R-FtsZ complemented for its wild type counterpart as observed by a complementation test using JKD7-1/pKD3 cells. However, the bacterial cells became elongated upon overexpression of the mutant allele. We modeled the structure of E93R-FtsZ using the structures of MtbFtsZ/Methanococcus jannaschi FtsZ (MjFtsZ) dimers as templates. The MtbFtsZ-based structure suggests that the Arg(93)-Glu(138) salt bridge provides the additional stability, whereas the effect of mutation appears to be indirect (allosteric) if the EcFtsZ dimer is similar to that of MjFtsZ. The data presented in this study suggest that an increase in the stability of the FtsZ protofilaments is detrimental for the bacterial cytokinesis.

Fermentation with transketolase (tkt) deficient strain of Bacillus is the only reported industrially viable process for production of D-ribose, a commercially important pentose sugar. In addition to direct effects of tkt deficiency, the mutation in non-oxidative part of pentose phosphate pathway (PPP) is known to display several unexpected physiological characteristics such as decreased ability to utilize D-glucose, altered carbon catabolite repression, lack of motility, etc. Here we demonstrate the morphological plasticity of tkt deficient strain of Bacillus pumilus ATCC 21951 and its possible relation with D-ribose productivity, a measure of carbon flux through PPP. The bacilli divide normally in nutrient rich media such as Luria-Bertani (LB) broth while showing cell elongation of up to 20-fold without a visible septum accompanied by moderate to high extracellular D-ribose accumulation in glucose-rich media. The cells stained with DAPI (4'-6-diamidino-2-phenylindole) and anti FtsZ antibody showed nucleoid separation and Z-ring formation in LB broth but not in glucose-rich media. FtsZ protein is known to localize at the future division site forming a ring, called Z-ring, at an early stage in cytokinesis. The strain experiences inhibition or delay in Z-ring formation resulting in cell elongation, possibly due to its altered cell membrane composition resulting from tkt deficiency. We hypothesize that the lack of PPP intermediates may have two effects on the strain: (i) altered the cell membrane leading to delay in Z-ring formation and cell elongation; and (ii) induction of genes of the oxidative part of PPP resulting in D-ribose accumulation. Nutrient rich media such as LB broth may alleviate these metabolite deficiencies thereby restoring normal cell division and inhibiting excessive D-ribose accumulation. The D-ribose productivity and cell elongation may therefore be co-morbid. The results have implications in designing optimal media and monitoring strategy based on morphological analysis.

Precise control of actin filament length is essential to many cellular processes. Formins processively elongate filaments, whereas capping protein (CP) binds to barbed ends and arrests polymerization. While genetic and biochemical evidence has indicated that these two proteins function antagonistically, the mechanism underlying the antagonism has remained unresolved. Here we use multi-wavelength single-molecule fluorescence microscopy to observe the fully reversible formation of a long-lived ‘decision complex' in which a CP dimer and a dimer of the formin mDia1 simultaneously bind the barbed end. Further, mDia1 displaced from the barbed end by CP can randomly slide along the filament and later return to the barbed end to re-form the complex. Quantitative kinetic analysis reveals that the CP-mDia1 antagonism that we observe in vitro occurs through the decision complex. Our observations suggest new molecular mechanisms for the control of actin filament length and for the capture of filament barbed ends in cells., Formins promote the polymerization of actin filaments at barbed ends and capping protein prevents polymerization. Here the authors use single molecule imaging to directly visualize a decision complex consisting of the formin mDia1 and capping protein bound simultaneously to the filament barbed end and the dynamic mechanisms by which it forms and dissociates.

BACKGROUND: Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. RESULTS: Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. CONCLUSION: The mechanism of actin assembly is highly conserved over evolution. SIGNIFICANCE: APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla.

The assembly of FtsZ is considered to be a fundamental process during the bacterial cytokinesis. We used several complimentary techniques to probe the assembly of recombinant Escherichia coli FtsZ (EcFtsZ) and Mycobacterium tuberculosis FtsZ (MtbFtsZ) proteins in vitro. As documented earlier, EcFtsZ was found to polymerize at much faster rate than MtbFtsZ. Interestingly, we found that MtbFtsZ produced higher sedimentable polymerized mass than that of the EcFtsZ and that MtbFtsZ formed thicker protofilaments than that of the EcFtsZ. The results indicated that the EcFtsZ polymers are more labile than the MtbFtsZ polymers. Further, divalent calcium exerted strikingly different effects on the assembly of EcFtsZ and MtbFtsZ. Divalent calcium strongly enhanced the assembly of EcFtsZ and promoted bundling and stability of the protofilaments. In contrast, it had no detectable effect on the assembly of MtbFtsZ. In vitro, divalent calcium bound to EcFtsZ with much stronger affinity than to MtbFtsZ and significantly affected the secondary structure of EcFtsZ whereas it did not cause any detectable change in the secondary structure of MtbFtsZ. The results suggested that the assembly characteristics of EcFtsZ and MtbFtsZ are different and indicated that the assembly dynamics of these proteins are regulated by different mechanisms.

The assembly of FtsZ plays an important role in bacterial cell division. Mycobacterium tuberculosis FtsZ (MtbFtsZ) has a single cysteine residue at position 155. We have investigated the role of the lone cysteine residue in the assembly of MtbFtsZ using different complimentary approaches, namely chemical modification by a thiol-specific reagent 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or a cysteine-chelating agent HgCl(2), and site-directed mutagenesis of the cysteine residue. HgCl(2) strongly reduced the polymerized mass of MtbFtsZ while it had no detectable effect on the polymerization of Escherichia coli FtsZ, which lacks a cysteine residue. HgCl(2) inhibited the protofilamentous assembly of MtbFtsZ and induced the aggregation of the protein. Further, HgCl(2) perturbed the secondary structure of MtbFtsZ and increased the binding of a hydrophobic probe 1-anilinonaphthalene-8-sulfonic acid (ANS) with MtbFtsZ, indicating that the binding of HgCl(2) altered the conformation of MtbFtsZ. Chemical modification of MtbFtsZ by DTNB also decreased the polymerized mass of MtbFtsZ. Further, the mutagenesis of Cys-155 to alanine caused a strong reduction in the assembly of MtbFtsZ. Under assembly conditions, the mutated protein formed aggregates instead of protofilaments. Far-UV CD spectroscopy and ANS binding suggested that the mutated MtbFtsZ has different conformation than that of the native MtbFtsZ. The effect of the mutation or chemical modification of Cys-155 on the MtbFtsZ assembly has been explained considering its location in the MtbFtsZ crystal structure. The results together suggest that the cysteine residue (Cys-155) of MtbFtsZ plays an important role in the assembly of MtbFtsZ into protofilaments.

Totarol, a diterpenoid phenol, has been shown to inhibit the proliferation of several pathogenic Gram-positive bacteria including Mycobacterium tuberculosis. In this study, totarol was found to inhibit the proliferation of Bacillus subtilis cells with a minimum inhibitory concentration of 2 microM. It did not detectably perturb the membrane structure of B. subtilis; it strongly induced the filamentation in B. subtilis cells, suggesting that it inhibits bacterial cytokinesis. Totarol (1.5 microM) reduced the frequency of the Z-ring occurrence per micrometer of the bacterial cell length but did not affect the nucleoid frequency, suggesting that it blocks cytokinesis by inhibiting the formation of the Z-ring. The assembly dynamics of FtsZ is thought to play an important role in the formation and functioning of the Z-ring, a machine that engineers cytokinesis in bacteria. Since totarol was shown to inhibit the proliferation of M. tuberculosis, we examined the effects of totarol on the assembly dynamics of M. tuberculosis FtsZ (MtbFtsZ) in vitro. Totarol decreased the assembly of MtbFtsZ protofilaments and potently suppressed the GTPase activity of MtbFtsZ. It bound to MtbFtsZ with a dissociation constant of 11 +/- 2.3 microM. It increased the fluorescence intensity of the MtbFtsZ-1-anilinonaphthalene-8-sulfonic acid complex and inhibited the fluorescence intensity of N-(1-pyrene)maleimide-labeled MtbFtsZ, suggesting that totarol induces conformational changes in MtbFtsZ. The results indicated that totarol can perturb the assembly dynamics of FtsZ protofilaments in the Z-ring. Totarol exhibited extremely weak inhibitory effects on HeLa cell proliferation. It did not affect microtubule organization in HeLa cells. The results suggest that totarol inhibits bacterial proliferation by targeting FtsZ and it may be useful as a lead compound to develop an effective antitubercular drug.

Metastasis of adenoid cystic carcinoma to the kidney is rare. We present a case of a 50 year old male with complaints and radiological findings suggestive of renal cell carcinoma and lung deposits. However, fine needle aspiration was suggestive of adenoid cystic carcinoma metastatic deposits. It was later elicited that the patient had undergone surgery for parotid tumour seven years back which on histology was an adenoid cystic carcinoma. The case is being presented for its rarity.