A Kinesin-14 Motor Activates Neocentromeres to Promote Meiotic Drive in Maize.

Maize abnormal chromosome 10 (Ab10) encodes a classic example of true meiotic drive that converts heterochromatic regions called knobs into motile neocentromeres that are preferentially transmitted to egg cells. Here, we identify a cluster of eight genes on Ab10, called the Kinesin driver (Kindr) complex, that are required for both neocentromere motility and preferential transmission. Two meiotic drive mutants that lack neocentromere activity proved to be kindr epimutants with increased DNA methylation across the entire gene cluster. RNAi of Kindr induced a third epimutant and corresponding loss of meiotic drive. Kinesin gliding assays and immunolocalization revealed that KINDR is a functional minus-end-directed kinesin that localizes specifically to knobs containing 180 bp repeats. Sequence comparisons suggest that Kindr diverged from a Kinesin-14A ancestor ∼12 mya and has driven the accumulation of > 500 Mb of knob repeats and affected the segregation of thousands of genes linked to knobs on all 10 chromosomes.

Distinct kinesin motors drive two types of maize neocentromeres.

A maize chromosome variant called abnormal chromosome 10 (Ab10) converts knobs on chromosome arms into neocentromeres, causing their preferential segregation to egg cells in a process known as meiotic drive. We previously demonstrated that the gene Kinesin driver (Kindr) on Ab10 encodes a kinesin-14 required to mobilize neocentromeres made up of the major tandem repeat knob180. Here we describe a second kinesin-14 gene, TR-1 kinesin (Trkin), that is required to mobilize neocentromeres made up of the minor tandem repeat TR-1. Trkin lies in a 4-Mb region of Ab10 that is not syntenic with any other region of the maize genome and shows extraordinary sequence divergence from Kindr and other kinesins in plants. Despite its unusual structure, Trkin encodes a functional minus end-directed kinesin that specifically colocalizes with TR-1 in meiosis, forming long drawn out neocentromeres. TRKIN contains a nuclear localization signal and localizes to knobs earlier in prophase than KINDR. The fact that TR-1 repeats often co-occur with knob180 repeats suggests that the current role of the TRKIN/TR-1 system is to facilitate the meiotic drive of the KINDR/knob180 system.

Cholesterol in the cargo membrane amplifies tau inhibition of kinesin-1-based transport.

Intracellular cargos are often membrane-enclosed and transported by microtubule-based motors in the presence of microtubule-associated proteins (MAPs). Whereas increasing evidence reveals how MAPs impact the interactions between motors and microtubules, critical questions remain about the impact of the cargo membrane on transport. Here we combined in vitro optical trapping with theoretical approaches to determine the effect of a lipid cargo membrane on kinesin-based transport in the presence of MAP tau. Our results demonstrate that attaching kinesin to a fluid lipid membrane reduces the inhibitory effect of tau on kinesin. Moreover, adding cholesterol, which reduces kinesin diffusion in the cargo membrane, amplifies the inhibitory effect of tau on kinesin binding in a dosage-dependent manner. We propose that reduction of kinesin diffusion in the cargo membrane underlies the effect of cholesterol on kinesin binding in the presence of tau, and we provide a simple model for this proposed mechanism. Our study establishes a direct link between cargo membrane cholesterol and MAP-based regulation of kinesin-1. The cholesterol effects uncovered here may more broadly extend to other lipid alterations that impact motor diffusion in the cargo membrane, including those associated with aging and neurological diseases.

In Utero and Childhood/Adolescence Exposure to Tobacco Smoke, Genetic Risk, and Lung Cancer Incidence and Mortality in Adulthood.

Rationale: The individual effects of early-life tobacco smoke exposure and its interactions with genetic factors on lung cancer in adulthood remain unclear. Objectives: To investigate the associations of early-life tobacco exposures as well as their interactions with polygenic risk scores (PRSs) with lung cancer incidence and mortality. Methods: A total of 432,831 participants from the UK Biobank study were included. We estimated the associations of in utero exposure to tobacco smoke, the age of smoking initiation and their interactions with PRSs with lung cancer incidence and mortality in adulthood using Cox proportional hazard models. Measurements and Main Results: Lung cancer incidence (hazard ratio [HR]: 1.59, 95% confidence interval [CI], 1.44-1.76) increased among participants with in utero tobacco exposure. Multivariable-adjusted HRs (with 95% CIs) of lung cancer incidence for smoking initiation in adulthood, adolescence, and childhood (versus never-smokers) were 6.10 (5.25-7.09), 9.56 (8.31-11.00), and 15.15 (12.90-17.79) (Ptrend < 0.001). Similar findings were observed in lung cancer mortality. Participants with high PRSs and in utero tobacco exposure (versus low PRSs participants without in utero exposure) had an HR of 2.35 for lung cancer incidence (95% CI, 1.97-2.80, Pinteraction = 0.089) and 2.43 for mortality (95% CI, 2.05-2.88, Pinteraction = 0.032). High PRSs with smoking initiation in childhood (versus never-smokers with low PRSs) had HRs of 18.71 for incidence (95% CI, 14.21-24.63, Pinteraction = 0.004) and 19.74 for mortality (95% CI, 14.98-26.01, Pinteraction = 0.033). Conclusions: In utero and childhood/adolescence exposure to tobacco smoke and its interaction with genetic factors may substantially increase the risks of lung cancer incidence and mortality in adulthood.

Effects of apparent temperature on daily outpatient and inpatient visits for cause-specific respiratory diseases in Ganzhou, China: a time series study.

BACKGROUND: Non-optimum temperatures are associated with increased risk of respiratory diseases, but the effects of apparent temperature (AT) on respiratory diseases remain to be investigated. METHODS: Using daily data from 2016 to 2020 in Ganzhou, a large city in southern China, we analyzed the impact of AT on outpatient and inpatient visits for respiratory diseases. We considered total respiratory diseases and five subtypes (influenza and pneumonia, upper respiratory tract infection (URTI), lower respiratory tract infection (LRTI), asthma and chronic obstructive pulmonary disease [COPD]). Our analysis employed a distributed lag nonlinear model (DLNM) combined with a generalized additive model (GAM). RESULTS: We recorded 94,952 outpatients and 72,410 inpatients for respiratory diseases. We found AT significantly non-linearly associated with daily outpatient and inpatient visits for total respiratory diseases, influenza and pneumonia, and URTI, primarily during comfortable AT levels, while it was exclusively related with daily inpatient visits for LRTI and COPD. Moderate heat (32.1 °C, the 75.0th centile) was observed with a significant effect on both daily outpatient and inpatient visits for total respiratory diseases at a relative risk of 1.561 (1.161, 2.098) and 1.276 (1.027, 1.585), respectively (both P < 0.05), while the results of inpatients became insignificant with the adjustment for CO and O3. The attributable fractions in outpatients and inpatients were as follows: total respiratory diseases (24.43% and 18.69%), influenza and pneumonia (31.54% and 17.33%), URTI (23.03% and 32.91%), LRTI (37.49% and 30.00%), asthma (9.83% and 3.39%), and COPD (30.67% and 10.65%). Stratified analyses showed that children ≤5 years old were more susceptible to moderate heat than older participants. CONCLUSIONS: In conclusion, our results indicated moderate heat increase the risk of daily outpatient and inpatient visits for respiratory diseases, especially among children under the age of 5.

Acrolein Exposure Impaired Glucose Homeostasis and Increased Risk of Type 2 Diabetes: An Urban Adult Population-Based Cohort Study with Repeated Measures.

Acrolein is an identified high-priority hazardous air pollutant ubiquitous in daily life and associated with cardiometabolic risk that attracts worldwide attention. However, the etiology role of acrolein exposure in glucose dyshomeostasis and type 2 diabetes (T2D) is unclear. This repeated-measurement prospective cohort study included 3522 urban adults. Urine/blood samples were repeatedly collected for determinations of acrolein metabolites (N-acetyl-S-(3-hydroxypropyl)-l-cysteine, N-acetyl-S-(2-carboxyethyl)-l-cysteine; acrolein exposure biomarkers), glucose homeostasis, and T2D at baseline and a three-year follow-up. We found that each 3-fold increment in acrolein metabolites was cross-sectionally associated with 5.91-6.52% decrement in homeostasis model assessment-insulin sensitivity (HOMA-IS) and 0.07-0.14 mmol/L, 4.02-4.57, 5.91-6.52, 19-20, 18-19, and 23-31% increments in fasting glucose (FPG), fasting insulin (FPI), HOMA-insulin resistance (HOMA-IR), risks of prevalent IR, impaired fasting glucose (IFG), and T2D, respectively; longitudinally, participants with sustained-high acrolein metabolite levels had increased risks of incident IR, IFG, and T2D by 63-80, 87-99, and 120-154%, respectively (P < 0.05). In addition, biomarkers of heme oxygenase-1 activity (exhaled carbon monoxide), lipid peroxidation (8-iso-prostaglandin-F2α), protein carbonylation (protein carbonyls), and oxidative DNA damage (8-hydroxy-deoxyguanosine) mediated 5.00-38.96% of these associations. Our study revealed that acrolein exposure may impair glucose homeostasis and increase T2D risk via mediating mechanisms of heme oxygenase-1 activation, lipid peroxidation, protein carbonylation, and oxidative DNA damage.

Short-term impacts of air pollution on the platelet-lymphocyte ratio and neutrophil-lymphocyte ratio among urban adults in China.

The short-term impacts of urban air pollution on the platelet-lymphocyte ratio (PLR) and neutrophil-lymphocyte ratio (NLR) remain obscure. In this study, we included 3487 urban adults from the Wuhan-Zhuhai cohort. Individual inhalation exposure to air pollutants was estimated by combining participants' daily breath volume and ambient concentrations of six air pollutants (including fine particulate matter (PM2.5), inhalable particulate matter (PM10), nitrogen dioxide (NO2), sulfur dioxide (SO2), carbon monoxide (CO) and ozone (O3)). The cumulative impacts were assessed by applying lag structures of up to 7 days before the survey date. Associations of air pollutants with PLR and NLR were assessed using a linear mixed model and Bayesian kernel machine regression (BKMR) model. We found that PLR was negatively related to PM2.5 (lag02-lag06), PM10 (lag02-lag07), NO2 (lag02-lag07), and SO2 (lag03-lag05) and NLR was negatively related to PM10 (lag05 and lag07). In the BKMR model, a negative joint association between the six-air-pollutant mixture and PLR and NLR was observed, whereas PM10 and NO2 appeared to be more important than the other pollutants in the mixture. The negative impact of air pollutants was stronger in males, participants with lower body mass index (< 24 kg/m2), those cooking meals at home, drinkers, and non-exercisers. In conclusion, short-term exposure to air pollutants is significantly related to PLR and NLR in peripheral blood. PLR and NLR may provide new insight into the molecular mechanism underlying the adverse health impact of air pollutants.

The global burden of disease attributable to high fasting plasma glucose in 204 countries and territories, 1990-2019: An updated analysis for the Global Burden of Disease Study 2019.

AIMS: High fasting plasma glucose (HFPG) is an independent risk factor for several adverse health outcomes and has become a serious public health problem. We aimed to evaluate the spatial pattern and temporal trend of disease burden attributed to HFPG from 1990 to 2019 using data from the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2019. MATERIALS AND METHODS: Using data from GBD 2019, we estimated the numbers and age-standardized rates of deaths and disability-adjusted life years (DALYs) attributed to HFPG by calendar year, age, gender, country, region, Socio-demographic Index (SDI), and specific causes. The joinpoint regression analysis was used to assess the temporal trends of deaths and DALYs from 1990 to 2019. RESULTS: In 2019, globally, the numbers of deaths and DALYs attributable to HFPG were approximately 6.50 million and 172.07 million, respectively, with age-standardized rates of 83.00 per 100,000 people and 2104.26 per 100,000 people, respectively. From 1990 to 2019, the global numbers of deaths and DALYs attributed to HFPG have over doubled. The age-standardized rate of DALYs showed an increasing trend, particularly in males and in regions with middle SDI or below. The leading causes of the global disease burden attributable to HFPG in 2019 were diabetes mellitus, ischaemic heart disease, stroke, and chronic kidney disease. CONCLUSIONS: HFPG is an important contributor to increasing the global and regional disease burden. Necessary measures should be taken to curb the growing burden attributed to HFPG, particularly in males and in regions with middle SDI or below.

The association between serum albumin and depression in chronic liver disease may differ by liver histology.

BACKGROUND: There are conflicting results regarding the association between chronic liver disease (CLD) and depression and the underlying biological mechanisms are lack of investigation. To address the impact of depression and its effects on the management of CLD, its biological marker is critical to be identified. The present study explored the association between serum albumin and depression in CLD patients and whether the association varied in different liver histological stages. METHODS: Based on the United States National Health and Nutrition Examination Survey 2017-2018, the data of serum albumin and depressive symptoms from 627 participants with CLD were used. Depression symptoms were assessed with the nine-item Patient Health Questionnaire (PHQ-9). We used multivariate linear regression to evaluate the association between serum albumin and PHQ-9 scores. Stratified analysis was performed according to the liver histology examined by vibration controlled transient elastography. RESULTS: Serum albumin level was inversely associated with PHQ-9 scores in the multivariate regression model after adjusting for mainly potential confounders (ß = - 1.113, 95% CI: - 2.065 to - 0.162, P = 0.0221). In the subgroup analysis stratified by gender, controlled attenuation parameter (CAP) and liver stiffness measurement (LSM), the inverse association remained significant in female (ß = - 2.002, 95% CI: - 3.515 to - 0.489, P = 0.0100), patients with CAP < 274 dB/m (ß = - 2.215, 95% CI: - 3.621 to - 0.808, P = 0.0023) and patients with LSM ≥8.2 kPa (ß = - 4.074, 95% CI: - 6.237 to - 1.911, P = 0.0003). Moreover, the association was much stronger when the serum albumin was higher than 3.4 g/dL among patients with LSM ≥8.2 kPa (ß = - 4.835, 95% CI: - 7.137 to - 2.533, P < 0.0001). CONCLUSION: Our study revealed an inverse association between serum albumin and depression in CLD patients and this association differed according to liver histological changes. Serum albumin could be a warning marker for depressive symptoms in CLD patients. It is essential for taking corresponding intervention strategies.

Regional Alteration within the Cerebellum and the Reorganization of the Cerebrocerebellar System following Poststroke Aphasia.

Recently, an increasing number of studies have highlighted the role of the cerebellum in language processing. However, the role of neural reorganization within the cerebellum as well as within the cerebrocerebellar system caused by poststroke aphasia remains unknown. To solve this problem, in the present study, we investigated regional alterations of the cerebellum as well as the functional reorganization of the cerebrocerebellar circuit by combining structural and resting-state functional magnetic resonance imaging (fMRI) techniques. Twenty patients diagnosed with aphasia following left-hemispheric stroke and 20 age-matched healthy controls (HCs) were recruited in this study. The Western Aphasia Battery (WAB) test was used to assess the participants' language ability. Gray matter volume, spontaneous brain activity, functional connectivity, and effective connectivity were examined in each participant. We discovered that gray matter volumes in right cerebellar lobule VI and right Crus I were significantly lower in the patient group, and the brain activity within these regions was significantly correlated with WAB scores. We also discovered decreased functional connectivity within the crossed cerebrocerebellar circuit, which was significantly correlated with WAB scores. Moreover, altered information flow between the cerebellum and the contralateral cerebrum was found. Together, our findings provide evidence for regional alterations within the cerebellum and the reorganization of the cerebrocerebellar system following poststroke aphasia and highlight the important role of the cerebellum in language processing within aphasic individuals after stroke.

Reliability and diagnostic accuracy of corrected slack angle derived from 2D-SWE in quantitating muscle spasticity of stroke patients.

BACKGROUND: To explore the feasibility of corrected slack angle acquired from two-dimensional shear wave elastography (2D-SWE) for quantitating the spasticity of medial gastrocnemius (MG) in stroke patients. METHODS: Consecutive stroke patients with spastic MG and matched healthy controls were recruited. Intra- and interobserver reliability of 2D-SWE measurement were evaluated, and the correlation between corrected slack angle and modified Ashworth scale (MAS) score was examined. The corrected slack angle before and after botulinum toxin A (BoNT-A) injection was compared and its diagnostic performance in classifying the severity of spasticity were assessed with receiver operating characteristic (ROC) curve analysis. RESULTS: The intra- (0.791 95% CI 0.432-0.932) and interobserver (0.751 95% CI 0.382-0.916) reliability of slack angle acquired with 2D-SWE were good. Significant correlation was found between corrected slack angle and MAS score (R = - 0.849, p < 0.001). The corrected slack angle increased after BoNT-A injection. The cutoff value of MAS ≥ 3 had the highest sensitivity (100%) and specificity (93.33%). The positive predictive value (PPV) for classification of MAS ≥ 1+ and the negative predictive value (NPV) for classification of MAS ≥ 3 were greater than 90%. CONCLUSION: 2D-SWE was a reliable method to quantitate the post-stroke spasticity. The corrected slack angle had advantage in classifying the severity of spasticity, especially in early identification of mild spasticity and confirmation of severe spasticity.

Effect of public health interventions on COVID-19 cases: an observational study.

BACKGROUND: As the epidemic of COVID-19 is gradually controlled in China, a summary of epidemiological characteristics and interventions may help control its global spread. METHODS: Data for COVID-19 cases in Hubei Province (capital, Wuhan) was extracted until 7 March 2020. The spatiotemporal distribution of the epidemic in four periods (before 10 January, 10-22 January, 23 January-6 February and 7 February-7 March) was evaluated, and the impacts of interventions were observed. RESULTS: Among 67 706 COVID-19 cases, 52 111 (76.97%) were aged 30-69 years old, and 34 680 (51.22%) were women. The average daily attack rates (95% CI) were 0.5 (0.3 to 0.7), 14.2 (13.2 to 15.1), 45.7 (44.0 to 47.5) and 8.6 (7.8 to 9.3) cases per 106 people in four periods, and the harmonic means (95% CI) of doubling times were 4.28 (4.01 to 4.55), 3.87 (3.78 to 3.98), 5.40 (4.83 to 6.05) and 45.56 (39.70 to 52.80) days. Compared with the first period, daily attack rates rose rapidly in the second period. In the third period, 14 days after 23 January, the daily average attack rate in and outside Wuhan declined by 33.8% and 48.0%; the doubling times increased by 95.0% and 133.2%. In the four periods, 14 days after 7 February, the daily average attack rate in and outside Wuhan decreased by 79.1% and 95.2%; the doubling times increased by 79.2% and 152.0%. CONCLUSIONS: The public health interventions were associated with a reduction in COVID-19 cases in Hubei Province, especially in districts outside of Wuhan.

YBX has functional roles in CpG-ODN against cold stress and bacterial infection of Misgurnus anguillicaudatus.

Misgurnus anguillicaudatus (M. anguillicaudatus) is a widely cultivated fish. However, in M. anguillicaudatus breeding, the frequent cold stress during daily breeding could induce immune suppression and increase the risk of infection, causing serious economic loss. Based on existing findings, CpG Oligonucleotides (CpG-ODNs) may be an ideal protective agent for low temperature fish breeding, performing anti-infective when faced with cold stress with cold shock proteins Y box binding proteins (YBX). Although YBX has pleiotropic functions, its roles in CpG-ODNs-mediated immunity (especially under cold situations) remain largely unexplored. To clarify the relationship among them, we identified the YBX1/YBX2 in M. anguillicaudatus and analyzed using a series of bioinformatics methods. After that, we immunized the fish with 3 types of CpG-ODNs and challenged with Aeromonas hydrophila (A. hydrophila). Here we showed that the best anti-bacterial effect of CpG-B was accompanied by the significant upregulation of YBX1. And the detection of the YBX1 downstream effectors confirmed that CpG-B induced the YBX1-mediated Th1 oriented responses to A. hydrophila by regulation of the NLRP3 (Caspase-A/-B), IL-1ß, IL-12 and IFN-γ. Afterwards, we found that under cold stress, CpG-B can activate the NLRP3 and NF-κB pathways through YBX1, a key mediator of anti-A. hydrophila in CpG-B immunization. In this study, we demonstrated CpG-B protection against infection in low temperature, and its interaction with YBX1, expanded the research of CpG-ODN under cold stress, and provided a new CpG-ODN application for low temperature fish farming.

Overexpression of pEGF improved the gut protective function of Clostridium butyricum partly through STAT3 signal pathway.

Clostridium butyricum (C. butyricum) is a probiotic that could promote animal growth and protect gut health. So far, current studies mainly keep up with the basic biological functions of C. butyricum, missing the effective strategy to further improve its protective efficiency. A recent report about C. butyricum alleviating intestinal injury through epidermal growth factor receptor (EGFR) inspired us to bridge this gap by porcine epidermal growth factor (EGF) overexpression. Lacking a secretory overexpression system, we constructed the recombinant strains overexpressing pEGF in C. butyricum for the first time and obtained 4 recombinant strains for highly efficient secretion of pEGF (BC/pPD1, BC/pSPP, BC/pGHF, and BC/pDBD). Compared to the wild-type strain, we confirmed that the expression level ranges of the intestinal development-related genes (Claudin-1, GLUT-2, SUC, GLP2R, and EGFR) and anti-inflammation-related gene (IL-10) in IPECs were upregulated under recombinant strain stimulation, and the growth of Staphylococcus aureus and Salmonella typhimurium was significantly inhibited as well. Furthermore, a particular inhibitor (stattic) was used to block STAT3 tyrosine phosphorylation, resulting in the downregulation on antibacterial effect of recombinant strains. This study demonstrated that the secretory overexpression of pEGF in C. butyricum could upregulate the expression level of EGFR, consequently improving the intestinal protective functions of C. butyricum partly following STAT3 signal activation in IPECs and making it a positive loop. These findings on the overexpression strains pointed out a new direction for further development and utilization of C. butyricum. KEY POINTS: • By 12 signal peptide screening in silico, 4 pEGF overexpression strains of C. butyricum/pMTL82151-pEGF for highly efficient secretion of pEGF were generated for the first time. • The secretory overexpression of pEGF promoted the intestinal development, antimicrobial action, and anti-inflammatory function of C. butyricum. • The overexpressed pEGF upregulated the expression level of EGFR and further magnified the gut protective function of recombinant strains which in turn partly depended on STAT3 signal pathway in IPECs.

The air quality changes and related mortality benefits during the coronavirus disease 2019 pandemic in China: Results from a nationwide forecasting study.

Air quality changes during the coronavirus disease 2019 (COVID-19) pandemic in China has attracted increasing attention. However, more details in the changes, future air quality trends, and related death benefits on a national scale are still unclear. In this study, a total of 352 Chinese cities were included. We collected air pollutants (including fine particulate matter [PM2.5], inhalable particulate matter [PM10], nitrogen dioxide [NO2], and ozone [O3]) data for each city from January 2015 to July 2020. Convolutional neural network-quantile regression (CNN-QR) forecasting model was used to predict pollutants concentrations from February 2020 to January 2021 and the changes in air pollutants were compared. The relationships between the socioeconomic factors and the changes and the avoided mortality due to the changes were further estimated. We found sharp declines in all air pollutants from February 2020 to January 2021. Specifically, PM2.5, PM10, NO2, and O3 would drop by 3.86 µg/m3 (10.81%), 4.84 µg/m3 (7.65%), 0.55 µg/m3 (2.18%), and 3.14 µg/m3 (3.36%), respectively. The air quality changes were significantly related to many of the socioeconomic factors, including the size of built-up area, gross regional product, population density, gross regional product per capita, and secondary industry share. And the improved air quality would avoid a total of 7237 p.m.2.5-related deaths (95% confidence intervals [CI]: 4935, 9209), 9484 p.m.10-related deaths (95%CI: 5362, 13604), 4249 NO2-related deaths (95%CI: 3305, 5193), and 6424 O3-related deaths (95%CI: 3480, 9367), respectively. Our study shows that the interventions to control COVID-19 would improve air quality, which had significant relationships with some socioeconomic factors. Additionally, improved air quality would reduce the number of non-accidental deaths.

Evolution and Antigenic Drift of Influenza A (H7N9) Viruses, China, 2017-2019.

After a sharp decrease of influenza A(H7N9) virus in China in 2018, highly pathogenic H7N9 viruses re-emerged in 2019. These H7N9 variants exhibited a new predominant subclade and had been cocirculating at a low level in eastern and northeastern China. Several immune escape mutations and antigenic drift were observed in H7N9 variants.

Otoferlin is a multivalent calcium-sensitive scaffold linking SNAREs and calcium channels.

Sensory hair cells rely on otoferlin as the calcium sensor for exocytosis and encoding of sound preferentially over the neuronal calcium sensor synaptotagmin. Although it is established that synaptotagmin cannot rescue the otoferlin KO phenotype, the large size and low solubility of otoferlin have prohibited direct biochemical comparisons that could establish functional differences between these two proteins. To address this challenge, we have developed a single-molecule colocalization binding titration assay (smCoBRA) that can quantitatively characterize full-length otoferlin from mammalian cell lysate. Using smCoBRA, we found that, although both otoferlin and synaptotagmin bind membrane fusion SNARE proteins, only otoferlin interacts with the L-type calcium channel Cav1.3, showing a significant difference between the synaptic proteins. Furthermore, otoferlin was found capable of interacting with multiple SNARE and Cav1.3 proteins simultaneously, forming a heterooligomer complex. We also found that a deafness-causing missense mutation in otoferlin attenuates binding between otoferlin and Cav1.3, suggesting that deficiencies in this interaction may form the basis for otoferlin-related hearing loss. Based on our results, we propose a model in which otoferlin acts as a calcium-sensitive scaffolding protein, localizing SNARE proteins proximal to the calcium channel so as to synchronize calcium influx with membrane fusion. Our findings also provide a molecular-level explanation for the observation that synaptotagmin and otoferlin are not functionally redundant. This study also validates a generally applicable methodology for quantitatively characterizing large, multivalent membrane proteins.

The Orphan Kinesin PAKRP2 Achieves Processive Motility via a Noncanonical Stepping Mechanism.

Phragmoplast-associated kinesin-related protein 2 (PAKRP2) is an orphan kinesin in Arabidopsis thaliana that is thought to transport vesicles along phragmoplast microtubules for cell plate formation. Here, using single-molecule fluorescence microscopy, we show that PAKRP2 is the first orphan kinesin to exhibit processive plus-end-directed motility on single microtubules as individual homodimers. Our results show that PAKRP2 processivity is achieved despite having an exceptionally long (32 residues) neck linker. Furthermore, using high-resolution nanoparticle tracking, we find that PAKRP2 steps via a hand-over-hand mechanism that includes frequent side steps, a prolonged diffusional search of the tethered head, and tight coupling of the ATP hydrolysis cycle to the forward-stepping cycle. Interestingly, truncating the PAKRP2 neck linker to 14 residues decreases the run length of PAKRP2; thus, the long neck linker enhances the processive behavior. Based on the canonical model of kinesin stepping, such a long neck linker is expected to decrease the processivity and disrupt the coupling of ATP hydrolysis to forward stepping. Therefore, we conclude that PAKRP2 employs a noncanonical strategy for processive motility, wherein a long neck linker is coupled with a slow ATP hydrolysis rate to allow for an extended diffusional search during each step without sacrificing processivity or efficiency.

Mechanisms underlying the dual-mode regulation of microtubule dynamics by Kip3/kinesin-8.

The kinesin-8 family of microtubule motors plays a critical role in microtubule length control in cells. These motors have complex effects on microtubule dynamics: they destabilize growing microtubules yet stabilize shrinking microtubules. The budding yeast kinesin-8, Kip3, accumulates on plus ends of growing but not shrinking microtubules. Here we identify an essential role of the tail domain of Kip3 in mediating both its destabilizing and its stabilizing activities. The Kip3 tail promotes Kip3's accumulation at the plus ends and facilitates the destabilizing effect of Kip3. However, the Kip3 tail also inhibits microtubule shrinkage and is required for promoting microtubule rescue by Kip3. These effects of the tail domain are likely to be mediated by the tubulin- and microtubule-binding activities that we describe. We propose a concentration-dependent model for the coordination of the destabilizing and stabilizing activities of Kip3 and discuss its relevance to cellular microtubule organization.

Allogeneic bone marrow-derived mesenchymal stromal cells for hepatitis B virus-related acute-on-chronic liver failure: A randomized controlled trial.

Mortality from hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) is high due to limited treatment options. Preclinical and clinical investigations have proved that treatment with mesenchymal stromal cells (MSCs) is beneficial for recovery from liver injury. We hypothesized that the outcome of HBV-related ACLF would be improved by MSC treatment. From 2010 to 2013, 110 patients with HBV-related ACLF were enrolled in this open-label, nonblinded randomized controlled study. The control group (n = 54) was treated with standard medical therapy (SMT) only. The experimental group (n = 56) was infused weekly for 4 weeks with 1.0 to 10 × 105 cells/kg allogeneic bone marrow-derived MSCs and then followed for 24 weeks. The cumulated survival rate of the MSC group was 73.2% (95% confidence interval 61.6%-84.8%) versus 55.6% (95% confidence interval 42.3%-68.9%) for the SMT group (P = 0.03). There were no infusion-related side effects, but fever was more frequent in MSC compared to SMT patients during weeks 5-24 of follow-up. No carcinoma occurred in any trial patient in either group. Compared with the control group, allogeneic bone marrow-derived MSC treatment markedly improved clinical laboratory measurements, including serum total bilirubin and Model for End-Stage Liver Disease scores. The incidence of severe infection in the MSC group was much lower than that in the SMT group (16.1% versus 33.3%, P = 0.04). Mortality from multiple organ failure and severe infection was higher in the SMT group than in the MSC group (37.0% versus 17.9%, P = 0.02). CONCLUSION: Peripheral infusion of allogeneic bone marrow-derived MSCs is safe and convenient for patients with HBV-related ACLF and significantly increases the 24-week survival rate by improving liver function and decreasing the incidence of severe infections. (Hepatology 2017;66:209-219).