Role of the repeat expansion size in predicting age of onset and severity in RFC1 disease.

RFC1 disease, caused by biallelic repeat expansion in RFC1, is clinically heterogeneous in terms of age of onset, disease progression and phenotype. We investigated the role of the repeat size in influencing clinical variables in RFC1 disease. We also assessed the presence and role of meiotic and somatic instability of the repeat. In this study, we identified 553 patients carrying biallelic RFC1 expansions and measured the repeat expansion size in 392 cases. Pearson's coefficient was calculated to assess the correlation between the repeat size and age at disease onset. A Cox model with robust cluster standard errors was adopted to describe the effect of repeat size on age at disease onset, on age at onset of each individual symptoms, and on disease progression. A quasi-Poisson regression model was used to analyse the relationship between phenotype and repeat size. We performed multivariate linear regression to assess the association of the repeat size with the degree of cerebellar atrophy. Meiotic stability was assessed by Southern blotting on first-degree relatives of 27 probands. Finally, somatic instability was investigated by optical genome mapping on cerebellar and frontal cortex and unaffected peripheral tissue from four post-mortem cases. A larger repeat size of both smaller and larger allele was associated with an earlier age at neurological onset [smaller allele hazard ratio (HR) = 2.06, P < 0.001; larger allele HR = 1.53, P < 0.001] and with a higher hazard of developing disabling symptoms, such as dysarthria or dysphagia (smaller allele HR = 3.40, P < 0.001; larger allele HR = 1.71, P = 0.002) or loss of independent walking (smaller allele HR = 2.78, P < 0.001; larger allele HR = 1.60; P < 0.001) earlier in disease course. Patients with more complex phenotypes carried larger expansions [smaller allele: complex neuropathy rate ratio (RR) = 1.30, P = 0.003; cerebellar ataxia, neuropathy and vestibular areflexia syndrome (CANVAS) RR = 1.34, P < 0.001; larger allele: complex neuropathy RR = 1.33, P = 0.008; CANVAS RR = 1.31, P = 0.009]. Furthermore, larger repeat expansions in the smaller allele were associated with more pronounced cerebellar vermis atrophy (lobules I-V ß = -1.06, P < 0.001; lobules VI-VII ß = -0.34, P = 0.005). The repeat did not show significant instability during vertical transmission and across different tissues and brain regions. RFC1 repeat size, particularly of the smaller allele, is one of the determinants of variability in RFC1 disease and represents a key prognostic factor to predict disease onset, phenotype and severity. Assessing the repeat size is warranted as part of the diagnostic test for RFC1 expansion.

Bromodomain inhibition overcomes treatment resistance in distinct molecular subtypes of melanoma.

Therapy of BRAF-mutant melanoma with selective inhibitors of BRAF (BRAFi) and MEK (MEKi) represents a major clinical advance but acquired resistance to therapy has emerged as a key obstacle. To date, no clinical approaches successfully resensitize to BRAF/MEK inhibition. Here, we develop a therapeutic strategy for melanoma using bromosporine, a bromodomain inhibitor. Bromosporine (bromo) monotherapy produced significant anti-tumor effects against established melanoma cell lines and patient-derived xenografts (PDXs). Combinatorial therapy involving bromosporine and cobimetinib (bromo/cobi) showed synergistic anti-tumor effects in multiple BRAFi-resistant PDX models. The bromo/cobi combination was superior in vivo to standard BRAFi/MEKi therapy in the treatment-naive BRAF-mutant setting and to MEKi alone in the setting of immunotherapy-resistant NRAS- and NF1-mutant melanoma. RNA sequencing of xenografts treated with bromo/cobi revealed profound down-regulation of genes critical to cell division and mitotic progression. Bromo/cobi treatment resulted in marked DNA damage and cell-cycle arrest, resulting in induction of apoptosis. These studies introduce bromodomain inhibition, alone or combined with agents targeting the mitogen activated protein kinase pathway, as a rational therapeutic approach for melanoma refractory to standard targeted or immunotherapeutic approaches.

Lessons Learned from Public Health and State Prison Collaborations during COVID-19 Pandemic and Multifacility Tuberculosis Outbreak, Washington, USA.

The large COVID-19 outbreaks in prisons in the Washington (USA) State Department of Corrections (WADOC) system during 2020 highlighted the need for a new public health approach to prevent and control COVID-19 transmission in the system's 12 facilities. WADOC and the Washington State Department of Health (WADOH) responded by strengthening partnerships through dedicated corrections-focused public health staff, improving cross-agency outbreak response coordination, implementing and developing corrections-specific public health guidance, and establishing collaborative data systems. The preexisting partnerships and trust between WADOC and WADOH, strengthened during the COVID-19 response, laid the foundation for a collaborative response during late 2021 to the largest tuberculosis outbreak in Washington State in the past 20 years. We describe challenges of a multiagency collaboration during 2 outbreak responses, as well as approaches to address those challenges, and share lessons learned for future communicable disease outbreak responses in correctional settings.

Antibiotics, Sedatives, and Catecholamines Further Compromise Sepsis-Induced Immune Suppression in Peripheral Blood Mononuclear Cells.

OBJECTIVES: We hypothesized that the immunosuppressive effects associated with antibiotics, sedatives, and catecholamines amplify sepsis-associated immune suppression through mitochondrial dysfunction, and there is a cumulative effect when used in combination. We thus sought to determine the impact of the exemplar drugs ciprofloxacin, propofol, and norepinephrine, used alone and in combination, at clinically relevant concentrations, on the ex vivo functionality of peripheral blood mononuclear cells (PBMCs) drawn from healthy, infected, and septic individuals. DESIGN: In vitro/ex vivo investigation. SETTING: University laboratory. SUBJECTS: Healthy volunteers, infected (nonseptic) patients in the emergency department, and septic ICU patients. INTERVENTIONS: PBMCs were isolated from these subjects and treated with ciprofloxacin (100 µg/mL), propofol (50 µg/mL), norepinephrine (10 µg/mL), or all three drugs combined, with and without lipopolysaccharide (100 ng/mL) for 6 or 24 hours. Comparison was made between study groups and against untreated cells. Measurements were made of cell viability, cytokine production, phagocytosis, human leukocyte antigen-DR (HLA-DR) status, mitochondrial membrane potential, mitochondrial reactive oxygen species production, and oxygen consumption. Gene expression in immune and metabolic pathways was investigated in PBMCs sampled from healthy volunteers coincubated with septic serum. MEASUREMENTS AND RESULTS: Coincubation with each of the drugs reduced cytokine production and phagocytosis in PBMCs isolated from septic patients, and healthy volunteers coincubated with septic serum. No effect was seen on HLA-DR surface expression. No cumulative effects were seen with the drug combination. Sepsis-induced changes in gene expression and mitochondrial functionality were not further affected by addition of any of the drugs. CONCLUSION: Drugs commonly used in critical care lead to significant immune dysfunction ex vivo and enhance sepsis-associated immunosuppression. Further studies are required to identify underlying mechanisms and potential impact on patient outcomes.

Pelvic floor injury during vaginal birth is life-altering and preventable: what can we do about it?

Pelvic floor disorders after childbirth have distressing lifelong consequences for women, requiring more than 300,000 women to have surgery annually. This represents approximately 10% of the 3 million women who give birth vaginally each year. Vaginal birth is the largest modifiable risk factor for prolapse, the pelvic floor disorder most strongly associated with birth, and is an important contributor to stress incontinence. These disorders require 10 times as many operations as anal sphincter injuries. Imaging shows that injuries of the levator ani muscle, perineal body, and membrane occur in up to 19% of primiparous women. During birth, the levator muscle and birth canal tissues must stretch to more than 3 times their original length; it is this overstretching that is responsible for the muscle tear visible on imaging rather than compression or neuropathy. The injury is present in 55% of women with prolapse later in life, with an odds ratio of 7.3, compared with women with normal support. In addition, levator damage can affect other aspects of hiatal closure, such as the perineal body and membrane. These injuries are associated with an enlarged urogenital hiatus, now known as antedate prolapse, and with prolapse surgery failure. Risk factors for levator injury are multifactorial and include forceps delivery, occiput posterior birth, older maternal age, long second stage of labor, and birthweight of >4000 g. Delivery with a vacuum device is associated with reduced levator damage. Other steps that might logically reduce injuries include manual rotation from occiput posterior to occiput anterior, slow gradual delivery, perineal massage or compresses, and early induction of labor, but these require study to document protection. In addition, teaching women to avoid pushing against a contracted levator muscle would likely decrease injury risk by decreasing tension on the vulnerable muscle origin. Providing care for women who have experienced difficult deliveries can be enhanced with early recognition, physical therapy, and attention to recovery. It is only right that women be made aware of these risks during pregnancy. Educating women on the long-term pelvic floor sequelae of childbirth should be performed antenatally so that they can be empowered to make informed decisions about management decisions during labor.

Electrodiagnostic subtyping in Guillain-Barré syndrome patients in the International Guillain-Barré Outcome Study.

BACKGROUND AND PURPOSE: Various electrodiagnostic criteria have been developed in Guillain-Barré syndrome (GBS). Their performance in a broad representation of GBS patients has not been evaluated. Motor conduction data from the International GBS Outcome Study (IGOS) cohort were used to compare two widely used criterion sets and relate these to diagnostic amyotrophic lateral sclerosis criteria. METHODS: From the first 1500 patients in IGOS, nerve conduction studies from 1137 (75.8%) were available for the current study. These patients were classified according to nerve conduction studies criteria proposed by Hadden and Rajabally. RESULTS: Of the 1137 studies, 68.3% (N = 777) were classified identically according to criteria by Hadden and Rajabally: 111 (9.8%) axonal, 366 (32.2%) demyelinating, 195 (17.2%) equivocal, 35 (3.1%) inexcitable and 70 (6.2%) normal. Thus, 360 studies (31.7%) were classified differently. The areas of differences were as follows: 155 studies (13.6%) classified as demyelinating by Hadden and axonal by Rajabally; 122 studies (10.7%) classified as demyelinating by Hadden and equivocal by Rajabally; and 75 studies (6.6%) classified as equivocal by Hadden and axonal by Rajabally. Due to more strictly defined cutoffs fewer patients fulfilled demyelinating criteria by Rajabally than by Hadden, making more patients eligible for axonal or equivocal classification by Rajabally. In 234 (68.6%) axonal studies by Rajabally the revised El Escorial (amyotrophic lateral sclerosis) criteria were fulfilled; in axonal cases by Hadden this was 1.8%. CONCLUSIONS AND DISCUSSION: This study shows that electrodiagnosis in GBS is dependent on the criterion set utilized, both of which are based on expert opinion. Reappraisal of electrodiagnostic subtyping in GBS is warranted.

A Comparison of Functional Luminal Imaging Probe With High-resolution Manometry, Timed Barium Esophagram, and pH Impedance Testing to Evaluate Functional Luminal Imaging Probe's Diagnostic Capabilities.

OBJECTIVE: The aims of this study are to determine the functional luminal imaging probe's (FLIP) diagnostic utility by comparing FLIP measurements with results from other esophageal evaluation standards. BACKGROUND: The FLIP is an esophageal evaluation technique performed at the time of endoscopy. Few studies have evaluated FLIP diagnostic capabilities compared with the established testing techniques, including high-resolution manometry (HRIM), time barium esophagram (TBE), and 24-hour impedance-pH monitoring. PATIENTS AND METHODS: A retrospective review was performed for 413 preintervention patients who underwent FLIP testing during endoscopy. Data from HRIM, 24-hour pH monitoring, and TBE were compared. RESULTS: Abnormal Distensibility Index (DI) was associated with abnormal integrated relaxation pressure (IRP; P = 0.003). Average DI was higher in patients with abnormal IRP (>15 mm Hg) when a hiatal hernia was present (P = 0.025). The total agreement between correlated diagnoses from FLIP and HRIM was 33.5%. DI was not associated with acid exposure time on pH monitoring. Agreement between FLIP and TBE was 49% with a sensitivity of 98.1% and a specificity of 36.5%. A 60 mL distension had a significantly lower detection rate than 40 mL and 50 mL for active peristalsis and was unaffected by pressure (P < 0.05). CONCLUSIONS: FLIP as an adjunct to HRIM is supported by strong metric correlation. FLIP was not correlated to pH monitoring findings, suggesting FLIP is not useful in reflux assessment. The agreement between FLIP and TBE was lower than in previous studies. Hiatal hernia impacted the normality between DI and IRP, not between FLIP and TBE. We suggest analyzing peristaltic patterns on panometry at all fill volumes to optimize detection.

Functional Anatomy of Urogenital Hiatus Closure: the Perineal Complex Triad Hypothesis.

INTRODUCTION: Urogenital hiatus enlargement is a critical factor associated with prolapse and operative failure. This study of the perineal complex was performed to understand how interactions among its three structures: the levator ani, perineal membrane, and perineal body-united by the vaginal fascia-work to maintain urogenital hiatus closure. METHODS: Magnetic resonance images from 30 healthy nulliparous women with 3D reconstruction of selected subjects were used to establish overall geometry. Connection points and lines of action were based on perineal dissection in 10 female cadavers (aged 22-86 years), cross sections of 4 female cadavers (aged 14-35 years), and histological sections (cadavers aged 16 and 21 years). RESULTS: The perineal membrane originates laterally from the ventral two thirds of the ischiopubic rami and attaches medially to the perineal body and vaginal wall. The levator ani attaches to the perineal membrane's cranial surface, vaginal fascia, and the perineal body. The levator line of action in 3D reconstruction is oriented so that the levator pulls the medial perineal membrane cranio-ventrally. In cadavers, simulated levator contraction and relaxation along this vector changes the length of the membrane and the antero-posterior diameter of the urogenital hiatus. Loss of the connection of the left and right perineal membranes through the perineal body results in diastasis of the levator and a widened hiatus, as well as a downward rotation of the perineal membrane. CONCLUSION: Interconnections involving the levator ani muscles, perineal membrane, perineal body, and vaginal fascia form the perineal complex surrounding the urogenital hiatus in an arrangement that maintains hiatal closure.

Predicting Leg Forces and Knee Moments Using Inertial Measurement Units: An In Vitro Study.

We compared the ability of seven machine learning algorithms to use wearable inertial measurement unit (IMU) data to identify the severe knee loading cycles known to induce microdamage associated with anterior cruciate ligament rupture. Sixteen cadaveric knee specimens, dissected free of skin and muscle, were mounted in a rig simulating standardized jump landings. One IMU was located above and the other below the knee, the applied three-dimensional action and reaction loads were measured via six-axis load cells, and the three-dimensional knee kinematics were also recorded by a laboratory motion capture system. Machine learning algorithms were used to predict the knee moments and the tibial and femur vertical forces; 13 knees were utilized for training each model, while three were used for testing its accuracy (i.e., normalized root-mean-square error) and reliability (Bland-Altman limits of agreement). The results showed the models predicted force and knee moment values with acceptable levels of error and, although several models exhibited some form of bias, acceptable reliability. Further research will be needed to determine whether these types of models can be modified to attenuate the inevitable in vivo soft tissue motion artifact associated with highly dynamic activities like jump landings.

Seroconversion and fever are dose-dependent in a nonhuman primate model of inhalational COVID-19.

While evidence exists supporting the potential for aerosol transmission of SARS-CoV-2, the infectious dose by inhalation remains unknown. In the present study, the probability of infection following inhalation of SARS-CoV-2 was dose-dependent in a nonhuman primate model of inhalational COVID-19. The median infectious dose, assessed by seroconversion, was 52 TCID50 (95% CI: 23-363 TCID50), and was significantly lower than the median dose for fever (256 TCID50, 95% CI: 102-603 TCID50), resulting in a group of animals that developed an immune response post-exposure but did not develop fever or other clinical signs of infection. In a subset of these animals, virus was detected in nasopharyngeal and/or oropharyngeal swabs, suggesting that infected animals without signs of disease are able to shed virus and may be infectious, which is consistent with reports of asymptomatic spread in human cases of COVID-19. These results suggest that differences in exposure dose may be a factor influencing disease presentation in humans, and reinforce the importance of public health measures that limit exposure dose, such as social distancing, masking, and increased ventilation. The dose-response data provided by this study are important to inform disease transmission and hazard modeling, and, ultimately, mitigation strategies. Additionally, these data will be useful to inform dose selection in future studies examining the efficacy of therapeutics and vaccines against inhalational COVID-19, and as a baseline in healthy, young adult animals for assessment of the importance of other factors, such as age, comorbidities, and viral variant, on the infectious dose and disease presentation.

A unified pelvic floor conceptual model for studying morphological changes with prolapse, age, and parity.

Several 2-dimensional and 3-dimensional measurements have been used to assess changes in pelvic floor structures and shape. These include assessment of urogenital and levator hiatus dimensions, levator injury grade, levator bowl volume, and levator plate shape. We argue that each assessment reflects underlying changes in an individual aspect of the overall changes in muscle and fascial structures. Vaginal delivery, aging, and interindividual variations in anatomy combine to affect pelvic floor structures and their connections in different ways. To date, there is no unifying conceptual model that permits the evaluation of how these many measures relate to one another or that reflects overall pelvic floor structure and function. Therefore, this study aimed to describe a unified pelvic floor conceptual model to better understand how the aforementioned changes to the pelvic floor structures and their biomechanical interactions affect pelvic organ support with vaginal birth, prolapse, and age. In this model, the pelvic floor is composed of 5 key anatomic structures: the (1) pubovisceral, (2) puborectal, and (3) iliococcygeal muscles with their superficial and inferior fascia; (4) the perineal membrane or body; and (5) the anal sphincter complex. Schematically, these structures are considered to originate from pelvic sidewall structures and meet medially at important connection points that include the anal sphincter complex, perineal body, and anococcygeal raphe. The pubovisceral muscle contributes primarily to urogenital hiatus closure, whereas the puborectal muscle is mainly related to levator hiatus closure, although each muscle contributes to the other. Dorsally and laterally, the iliococcygeal muscle forms a shelflike structure in women with normal support that spans the remaining area between these medial muscles and attachments to the pelvic sidewall. Other features include the levator plate, bowl volume, and anorectal angle. The pelvic floor conceptual model integrates existing observations and points out evident knowledge gaps in how parturition, injury, disease, and aging can contribute to changes associated with pelvic floor function caused by the detachment of one or more important connection points or pubovisceral muscle failure.

Two emerging phenotypes of atypical inclusion body myositis: illustrative cases.

OBJECTIVES: Sporadic inclusion body myositis (IBM) is the most common acquired myopathy in those aged above 50. It is classically heralded by weakness in the long finger flexors and quadriceps. The aim of this article is to describe five atypical cases of IBM, outlining two potential emerging clinical subsets of the disease. METHODS: We reviewed relevant clinical documentation and pertinent investigations for five patients with IBM. RESULTS: The first phenotype we describe is young-onset IBM in two patients who had symptoms since their early thirties. The literature supports that IBM can rarely present in this age range or younger. We describe a second phenotype in three middle-aged women who developed early bilateral facial weakness at presentation in tandem with dysphagia and bulbar impairment followed by respiratory failure requiring non-invasive ventilation (NIV). Within this group, two patients were noted to have macroglossia, another possible rare feature of IBM. CONCLUSIONS: Despite the classical phenotype described within the literature IBM can present in a heterogenous fashion. It is important to recognise IBM in younger patients and investigate for specific associations. The described pattern of facial diplegia, severe dysphagia, bulbar dysfunction and respiratory failure in female IBM patients requires further characterisation. Patients with this clinical pattern may require more complex and supportive management. Macroglossia is a potentially under recognised feature of IBM. The presence of macroglossia in IBM warrants further study, as its presence may lead to unnecessary investigations and delay diagnosis.

Hiatal failure: effects of pregnancy, delivery, and pelvic floor disorders on level III factors.

INTRODUCTION AND HYPOTHESIS: The failure of the levator hiatus (LH) and urogenital hiatus (UGH) to remain closed is not only associated with pelvic floor disorders, but also contributes to recurrence after surgical repair. Pregnancy and vaginal birth are key events affecting this closure. An understanding of normal and failed hiatal closure is necessary to understand, manage, and prevent pelvic floor disorders. METHODS: This narrative review was conducted by applying the keywords "levator hiatus" OR "genital hiatus" OR "urogenital hiatus" in PubMed. Articles that reported hiatal size related to pelvic floor disorders and pregnancy were chosen. Weighted averages for hiatal size were calculated for each clinical situation. RESULTS: Women with prolapse have a 22% and 30% larger LH area measured by ultrasound at rest and during Valsalva than parous women with normal support. Women with persistently enlarged UGH have 2-3 times higher postoperative failure rates after surgery for prolapse. During pregnancy, the LH area at Valsalva increases by 29% from the first to the third trimester in preparation for childbirth. The enlarged postpartum hiatus recovers over time, but does not return to nulliparous size after vaginal birth. Levator muscle injury during vaginal birth, especially forceps-assisted, is associated with increases in hiatal size; however, it only explains a portion of hiatus variation-the rest can be explained by pelvic muscle function and possibly injury to other level III structures. CONCLUSIONS: Failed hiatal closure is strongly related to pelvic floor disorders. Vaginal birth and levator injury are primary factors affecting this important mechanism.

Noninvasive ventilation in New Zealand: a national prevalence survey.

BACKGROUND: Home-based noninvasive ventilation (NIV) is an effective treatment for a range of conditions that cause respiratory failure which reduces hospitalisation and mortality and improves quality of life. AIMS: To collect NIV prevalence, disease burden and equity data needed for effective national NIV health service planning. METHODS: The authors collected demographics and the primary diagnosis of patients receiving publicly funded NIV in New Zealand in 2018 by surveying all providers. National and regional prevalence rates were calculated using adult population data (aged ≥20 years) for each District Health Board region compared with a 2011 study. A subanalysis of individual-level data was used to calculate age-standardised rates by diagnostic category. RESULTS: A total of 1197 adults were receiving NIV giving a national rate of 32.9 per 100 000; almost twice the 2011 rate (16.7 per 100 000). Significant regional variations in NIV provision (4.5-84.2 per 100 000) were observed. The most frequent indications were obesity hypoventilation syndrome (OHS) (562, 47%), obstructive pathologies (335, 28%) and neuromuscular disorders (175, 15%); all have significantly increased in prevalence since 2011. Maori and Pacific peoples were significantly overrepresented among NIV users (2.24 [95% confidence interval (CI), 1.72-2.93] and 7.03 [95% CI, 5.52-8.94], respectively). The prevalence of NIV-dependent use (>15 h/day) was 4%. CONCLUSIONS: Home-based NIV provision has doubled since the previous survey, reflecting increased burden from OHS and obstructive pathologies and a disproportionate disease burden among Maori and Pacific populations. The large regional variations are concerning and highlight the urgent requirement for national service specifications, education and equipment provision. Further research is needed to address access equity.

PHIP drives glioblastoma motility and invasion by regulating the focal adhesion complex.

The invasive behavior of glioblastoma is essential to its aggressive potential. Here, we show that pleckstrin homology domain interacting protein (PHIP), acting through effects on the force transduction layer of the focal adhesion complex, drives glioblastoma motility and invasion. Immunofluorescence analysis localized PHIP to the leading edge of glioblastoma cells, together with several focal adhesion proteins: vinculin (VCL), talin 1 (TLN1), integrin beta 1 (ITGB1), as well as phosphorylated forms of paxillin (pPXN) and focal adhesion kinase (pFAK). Confocal microscopy specifically localized PHIP to the force transduction layer, together with TLN1 and VCL. Immunoprecipitation revealed a physical interaction between PHIP and VCL. Targeted suppression of PHIP resulted in significant down-regulation of these focal adhesion proteins, along with zyxin (ZYX), and produced profoundly disorganized stress fibers. Live-cell imaging of glioblastoma cells overexpressing a ZYX-GFP construct demonstrated a role for PHIP in regulating focal adhesion dynamics. PHIP silencing significantly suppressed the migratory and invasive capacity of glioblastoma cells, partially restored following TLN1 or ZYX cDNA overexpression. PHIP knockdown produced substantial suppression of tumor growth upon intracranial implantation, as well as significantly reduced microvessel density and secreted VEGF levels. PHIP copy number was elevated in the classical glioblastoma subtype and correlated with elevated EGFR levels. These results demonstrate PHIP's role in regulating the actin cytoskeleton, focal adhesion dynamics, and tumor cell motility, and identify PHIP as a key driver of glioblastoma migration and invasion.

Comparative Analysis of microRNAs that Stratify in vitro Mammary stem and Progenitor Activity Reveals Functionality of Human miR-92b-3p.

Mammary stem/progenitor cells are fundamental for mammary gland development and function. However, much remains to be elucidated regarding their function in mammals beyond the traditionally studied rodents, human, and to a lesser extent, ruminants. Due to the growing appreciation for microRNAs (miRNAs) as regulators of stem cells and their progenitors, we compared miRNA expression in mammary stem/progenitor cells from mammals with varying mammary stem/progenitor activity in vitro, in order to identify miRNA candidates that regulate stem/progenitor self-renewal and function. Mammosphere-derived epithelial cells (MDECs), which are primary cell lines enriched in mammary stem and progenitor cells, were generated from six mammalian species (i.e., cow, human, pig, horse, dog, and rat) and small RNA sequencing was performed. We identified 9 miRNAs that were significantly differentially expressed in MDEC cultures with a low versus high mammary stem/progenitor activity. miR-92b-3p was selected for functional follow-up studies, as this miRNA is understudied in primary mammary cells but has well-described gene targets that are known to regulate mammary stem/progenitor activity. Altering the expression of miR-92b-3p in MDECs from species with low stem/progenitor activity (human and cow) and those with high stem/progenitor activity (dog and rat) via inhibition and overexpression, respectively, resulted in significantly decreased mammosphere formation of human MDECs, but showed no significant effects in cow, dog, or rat MDECs. This study is the first to perform small RNA sequencing in MDECs from various mammals and highlights that conserved miRNAs can have different functions in mammary stem/progenitor cells across species.

Induced mammary cancer in rat models: pathogenesis, genetics, and relevance to female breast cancer.

Mammary cancer, or breast cancer in women, is a polygenic disease with a complex etiopathogenesis. While much remains elusive regarding its origin, it is well established that chemical carcinogens and endogenous estrogens contribute significantly to the initiation and progression of this disease. Rats have been useful models to study induced mammary cancer. They develop mammary tumors with comparable histopathology to humans and exhibit differences in resistance or susceptibility to mammary cancer depending on strain. While some rat strains (e.g., Sprague-Dawley) readily form mammary tumors following treatment with the chemical carcinogen, 7,12-dimethylbenz[a]-anthracene (DMBA), other strains (e.g., Copenhagen) are resistant to DMBA-induced mammary carcinogenesis. Genetic linkage in inbred strains has identified strain-specific quantitative trait loci (QTLs) affecting mammary tumors, via mechanisms that act together to promote or attenuate, and include 24 QTLs controlling the outcome of chemical induction, 10 QTLs controlling the outcome of estrogen induction, and 4 QTLs controlling the outcome of irradiation induction. Moreover, and based on shared factors affecting mammary cancer etiopathogenesis between rats and humans, including orthologous risk regions between both species, rats have served as useful models for identifying methods for breast cancer prediction and treatment. These studies in rats, combined with alternative animal models that more closely mimic advanced stages of breast cancer and/or human lifestyles, will further improve our understanding of this complex disease.

The voltage-sensing domain of a hERG1 mutant is a cation-selective channel.

A cationic leak current known as an "omega current" may arise from mutations of the first charged residue in the S4 of the voltage sensor domains of sodium and potassium voltage-gated channels. The voltage-sensing domains (VSDs) in these mutated channels act as pores allowing nonspecific passage of cations, such as Li+, K+, Cs+, and guanidinium. Interestingly, no omega currents have been previously detected in the nonswapped voltage-gated potassium channels such as the human-ether-a-go-go-related (hERG1), hyperpolarization-activated cyclic nucleotide-gated, and ether-a-go-go channels. In this work, we discovered a novel omega current by mutating the first charged residue of the S4 of the hERG1, K525 to serine. To characterize this omega current, we used various probes, including the hERG1 pore domain blocker, dofetilide, to show that the omega current does not require cation flux via the canonical pore domain. In addition, the omega flux does not cross the conventional selectivity filter. We also show that the mutated channel (K525S hERG1) conducts guanidinium. These data are indicative of the formation of an omega current channel within the VSD. Using molecular dynamics simulations with replica-exchange umbrella sampling simulations of the wild-type hERG1 and the K525S hERG1, we explored the molecular underpinnings governing the cation flow in the VSD of the mutant. We also show that the wild-type hERG1 may form water crevices supported by the biophysical surface accessibility data. Overall, our multidisciplinary study demonstrates that the VSD of hERG1 may act as a cation-selective channel wherein a mutation of the first charged residue in the S4 generates an omega current. Our simulation uncovers the atomistic underpinning of this mechanism.

Whole-Genome Sequencing Surveillance and Machine Learning of the Electronic Health Record for Enhanced Healthcare Outbreak Detection.

BACKGROUND: Most hospitals use traditional infection prevention (IP) methods for outbreak detection. We developed the Enhanced Detection System for Healthcare-Associated Transmission (EDS-HAT), which combines whole-genome sequencing (WGS) surveillance and machine learning (ML) of the electronic health record (EHR) to identify undetected outbreaks and the responsible transmission routes, respectively. METHODS: We performed WGS surveillance of healthcare-associated bacterial pathogens from November 2016 to November 2018. EHR ML was used to identify the transmission routes for WGS-detected outbreaks, which were investigated by an IP expert. Potential infections prevented were estimated and compared with traditional IP practice during the same period. RESULTS: Of 3165 isolates, there were 2752 unique patient isolates in 99 clusters involving 297 (10.8%) patient isolates identified by WGS; clusters ranged from 2-14 patients. At least 1 transmission route was detected for 65.7% of clusters. During the same time, traditional IP investigation prompted WGS for 15 suspected outbreaks involving 133 patients, for which transmission events were identified for 5 (3.8%). If EDS-HAT had been running in real time, 25-63 transmissions could have been prevented. EDS-HAT was found to be cost-saving and more effective than traditional IP practice, with overall savings of $192 408-$692 532. CONCLUSIONS: EDS-HAT detected multiple outbreaks not identified using traditional IP methods, correctly identified the transmission routes for most outbreaks, and would save the hospital substantial costs. Traditional IP practice misidentified outbreaks for which transmission did not occur. WGS surveillance combined with EHR ML has the potential to save costs and enhance patient safety.

On the management of maternal pushing during the second stage of labor: a biomechanical study considering passive tissue fatigue damage accumulation.

BACKGROUND: During the second stage of labor, the maternal pelvic floor muscles undergo repetitive stretch loading as uterine contractions and strenuous maternal pushes combined to expel the fetus, and it is not uncommon that these muscles sustain a partial or complete rupture. It has recently been demonstrated that soft tissues, including the anterior cruciate ligament and connective tissue in sheep pelvic floor muscle, can accumulate damage under repetitive physiological (submaximal) loads. It is well known to material scientists that this damage accumulation can not only decrease tissue resistance to stretch but also result in a partial or complete structural failure. Thus, we wondered whether certain maternal pushing patterns (in terms of frequency and duration of each push) could increase the risk of excessive damage accumulation in the pelvic floor tissue, thereby inadvertently contributing to the development of pelvic floor muscle injury. OBJECTIVE: This study aimed to determine which labor management practices (spontaneous vs directed pushing) are less prone to accumulate damage in the pelvic floor muscles during the second stage of labor and find the optimum approach in terms of minimizing the risk of pelvic floor muscle injury. STUDY DESIGN: We developed a biomechanical model for the expulsive phase of the second stage of labor that includes the ability to measure the damage accumulation because of repetitive physiological submaximal loads. We performed 4 simulations of the second stage of labor, reflecting a directed pushing technique and 3 alternatives for spontaneous pushing. RESULTS: The finite element model predicted that the origin of the pubovisceral muscle accumulates the most damage and so it is the most likely place for a tear to develop. This result was independent of the pushing pattern. Performing 3 maternal pushes per contraction, with each push lasting 5 seconds, caused less damage and seemed the best approach. The directed pushing technique (3 pushes per contraction, with each push lasting 10 seconds) did not reduce the duration of the second stage of labor and caused higher damage accumulation. CONCLUSION: The frequency and duration of the maternal pushes influenced the damage accumulation in the passive tissues of the pelvic floor muscles, indicating that it can influence the prevalence of pelvic floor muscle injuries. Our results suggested that the maternal pushes should not last longer than 5 seconds and that the duration of active pushing is a better measurement than the total duration of the second stage of labor. Hopefully, this research will help to shed new light on the best practices needed to improve the experience of labor for women.