Mitf regulates gene expression networks implicated in B cell homeostasis, germinal center responses, and tolerance.

Introduction: The microphthalmia transcription factor Mitf has been shown to regulate B cell activation and tolerance. However, the underlying B cell-specific mechanisms responsible, and those that distinguish Mitf from closely related Mitf/TFE (MiT) transcription factors Tfe3, Tfeb, and Tfec, remain obscure. Methods: Two complementary mouse models of Mitf and MiT deficiency were used: the Mitfmi-vga9/mi-vga9 systemic loss-of-function mutation, and B-cell specific MiT family inactivation via transgenic expression of a trans-dominant negative (TDN) protein (TDN-B). These models were employed to identify MiT family candidate target genes and pathways. Results: Both models displayed spontaneous splenomegaly coincident with elevated plasma cell numbers, autoantibody titers, and proteinuria. These abnormalities appeared dependent on T helper cells, but independent of other non-B cell intrinsic effects of systemic Mitf inactivation. MiT inactivation in B cells augmented aspects of lupus-like autoimmune disease on the C57BL/6-Faslpr/lpr background. In both models, RNAseq of ex vivo resting B cells showed transcriptional upregulation of genes that control cell cycle, germinal center responses, and plasma cell differentiation. Among the genes strongly upregulated in both models were Socs6, Isp53 (Baiap1), S1pR2, and IgG2b/c. Mitf null B cells, but not TDN-B cells, showed evidence of type I interferon dysregulation. Discussion: These studies clarify Mitf's role as 1) a key regulator of a B cell intrinsic germinal center program that influences self-tolerance through novel target genes, and 2) a regulator of systemic inflammatory processes that can impact the B cell microenvironment. This distinction of Mitf's function from that of related MiT transcription factors advances our understanding of B cell regulation and autoimmunity.

A Procedural Checklist for Transurethral Resection of Bladder Tumors (TURBT) Enhances Operative Dictation and Assesses Surgeon Accuracy of Tumor Characteristic Predictions.

BACKGROUND: A lack of standardization is pervasive in procedural application and reporting templates for TURBT with the use of a surgical checklist proposed as a means for quality improvement. OBJECTIVE: To introduce a TURBT checklist to assess surgeon prediction accuracy and the impact of standardized documentation on quality of resection and oncologic outcomes. METHODS: Nine critical elements of a high-quality TURBT identified by literature review were incorporated into a prospectively implemented checklist for operative reports. The checklist included both visualized and predicted tumor characteristics. A retrospective single-institution analysis compared quality of dictation pre- and post-checklist implementation. Surgeon predictions were compared to final pathology reports to determine rates of concordance. Kaplan-Meier curves examined the association of checklist use with recurrence free survival (RFS). RESULTS: 333 operative reports were included in this analysis, of which 107 (32.1%) were completed pre-checklist implementation. The average number of critical elements reported was 8.69 with checklist use compared to 4.99 without (p < 0.001). There was no significant difference in RFS between the pre- and post-checklist cohorts (log-rank test p = 0.53). Surgeons were least and most accurate in predicting low grade tumor (43.5%) and absence of muscle invasion (96.6%), respectively. CONCLUSIONS: Incorporation of a TURBT surgical checklist improves operative dictation and quality of reporting but did not directly impact RFS. With quality of initial resection a proven correlate to recurrence rates, checklist implementation to improve surgical performance and long-term oncologic outcomes reveals an interesting area of exploration highlighting the need for more standardized methodology when performing these procedures.

Female Sexual Dysfunction as Measured by Low Sexual Frequency is Associated With Lower Socioeconomic Status: An Analysis of the National Health and Nutrition Examination Survey (NHANES), 2007-2016.

BACKGROUND: Female sexual dysfunction (FSD) is a complex disorder of biopsychosocial etiology, and FSD symptoms affect more than 40% of adult women worldwide. AIM: In this cross-sectional study, we sought to investigate the association between FSD and socioeconomic status (SES) in a nationally representative female adult population. METHODS: Economic and sexual data for women aged 20-59 from the 2007-2016 National Health and Nutrition Examination Survey, a United States nationwide representative database, was analyzed. Poverty income ratio (PIR), a ratio of family income to poverty threshold, was used as a measure of SES, and low sexual frequency was used as a measure of FSD. The association between FSD and SES was analyzed using survey-weighted logistic regression after adjusting for relevant social and gynecologic covariates, such as marital status and history of pregnancy, as well as significant medical comorbidities. OUTCOMES: We found that FSD, as measured by low sexual frequency, was associated with lower SES. RESULTS: Among the 7,348 women of mean age 38.4 (IQR 29-47) included in the final analysis, 26.3% of participants reported sexual frequency of 0-11 times/year and 73.7% participants reported sexual frequency >11 times/year. Participants of PIR <2 were 92% more likely to report sexual frequency ≤11 times/year than those of PIR ≥2 after adjusting for demographics, social history, gynecologic history and significant medical conditions (OR = 1.92; 95% CI = 1.21-3.05; P < .006). CLINICAL IMPLICATIONS: The evaluation and treatment of FSD may benefit from a comprehensive approach that takes SES into account. STRENGTHS & LIMITATIONS: This study is limited by its cross-sectional design, but it is strengthened by a large, nationally representative sample with extensive, standardized data ascertainment. CONCLUSION: Lower SES and lower sexual frequency are directly correlated among female adults in the United States; future studies should focus on social determinants of health as risk factors for FSD. Kim JI, Zhu D, Davila J, et al. Female Sexual Dysfunction as Measured by Low Sexual Frequency is Associated With Lower Socioeconomic Status: An Analysis of the National Health and Nutrition Examination Survey (NHANES), 2007-2016. J Sex Med 2022;19:90-97.

Clinical and epidemiological characterization in the follow-up of newborns with COVID-19: a descriptive study. / Caracterización clínica y epidemiológica en el seguimiento de recién nacidos con COVID-19: estudio descriptivo.

INTRODUCTION: COVID-19 disease affects newborns, but its middle and long-term effects are still unclear. OBJECTIVE: To describe the clinical and epidemiological characteristics and follow-up of newborns infected with SARS-CoV-2. METHODS: An observational and descriptive study. We included newborns with SARS-CoV-2 positive RT-PCR born from SARS-CoV-2 seropositive mothers. Delivery and newborn care were provided at the 'Instituto Nacional Materno Perinatal' from Peru between June 1 and September 30, 2020. Perinatal information was collected from medical records. Remote follow-up and face-to-face evaluations gathered epidemiological and clinical information, in addition to serological and RT-PCR tests for SARS-CoV-2. Descriptive statistics were used for analysis. RESULTS: During the study period, 4733 neonates were born at the institution. We found that 1488 (31.4%) were born from seropositive for SARS-CoV-2 mothers. Finally, we included the 34 (2.3%) newborns with positive RT-PCR for SARS-CoV-2. Regarding the included newborns, 29.4% were delivered by cesarean section, 26.5% had low birth weight, 11.8% were preterm, 26.5% were hospitalized, and one died. Twenty-eight had a remote follow-up, and 18 also had a face-to-face follow-up. A total of 64.3% were exclusively breastfed, 28.6% were mixed breastfed, and 7.1% used a substitute formula. The face-to-face evaluation was performed between one and four months of chronological age. We found that 100% had negative control RT-PCR test for COVID-19, 38.9% had a negative serological test (IgM, IgG), and 61.1% positive IgG. CONCLUSIONS: Neonatal SARS-CoV-2 infection is rare, and most infected infants are asymptomatic. Vaginal delivery, breastfeeding, and joint isolation did not related with complications during hospital care. Infants under remote and in-person follow-up showed favorable clinical evolution during the study period.

INTRODUCCIÓN: La enfermedad por COVID-19 ha sido reportada en recién nacidos; sin embargo, aún no son claros sus efectos en el seguimiento de neonatos. OBJETIVO: Describir las características clínicas, epidemiológicas y el seguimiento de recién nacidos infectados con SARS-CoV-2. MÉTODOS: Estudio observacional y descriptivo. Participaron recién nacidos que tuvieron PCR-TR positivo a SARS-CoV-2, hijos de madres seropositivas a SARS-CoV-2. La atención del parto y del recién nacido fueron en el Instituto Nacional Materno Perinatal de Perú, entre el 1 de junio y el 30 de septiembre de 2020. Se recogió información perinatal de registros médicos. Se realizó seguimiento remoto y evaluación presencial para descripción epidemiológica, clínica y resultados de pruebas serológicas y PCR-TR para SARS-CoV-2. En el análisis se usó estadística descriptiva. RESULTADOS: Durante el período de estudio nacieron 4733 recién nacidos. De estos niños, 1488 (31,4%) procedieron de gestantes seropositivas a SARS-CoV-2 y de ellos 34 (2,3%) tuvieron PCR-TR positivo a SARS-CoV-2. De los 34 recién nacidos 29,4% nació por cesárea, 26,5% tuvo bajo peso, 11,8% fue prematuro 26,5% tuvo indicación de hospitalización por patología y un neonato falleció. De los 34 neonatos, 28 tuvieron seguimiento remoto y de ellos 18 tuvieron además seguimiento presencial post alta. El 64,3% recibía lactancia materna exclusiva, 28,6% lactancia mixta y 7,1% usaba un sucedáneo. La evaluación presencial se realizó entre uno a cuatro meses de edad cronológica. El 100% tuvo prueba de PCR-TR de control para coronavirus negativa y 38,9% tuvo prueba serológica (IgM, IgG) negativa y 61,1% IgG positiva. CONCLUSIONES: La infección neonatal por SARS-CoV-2 es poco frecuente, la mayoría de infectados fueron asintomáticos. El parto vaginal, la lactancia materna y aislamiento conjunto no reportaron complicaciones en la evolución durante la atención hospitalaria. Los infantes en seguimiento remoto y presencial mostraron evolución clínica favorable durante el período de estudio.

Representation of Minority Groups in Key Pelvic Floor Disorder Trials.

BACKGROUND: Multicenter randomized clinical trials on pelvic floor disorders (PFDs) support evidence-based care. However, many of these studies include homogenous study populations lacking diversity. Heterogeneous sampling allows for greater generalizability while increasing knowledge regarding specific subgroups. The racial/ethnic makeup of key pelvic floor disorder (PFD) trials has not been examined. OBJECTIVE: This study aimed to investigate racial/ethnic representation in major PFD clinical trials in comparison to racial/ethnic distribution of PFD in the National Health and Nutritional Examination Survey (NHANES). METHODS: Demographic data were extracted from completed PFD Network (PFDN) and Urinary Incontinence Treatment Network studies, which have resulted in nearly 200 publications. Prevalence of PFD by race/ethnicity was obtained from the NHANES. A representative index (Observed "n" by PFD study/Expected "n" based on the NHANES-reported prevalence) was calculated as a measure of representation. Meta-analyses were performed for each outcome and overall with respect to race/ethnicity. RESULTS: Eighteen PFDN/Urinary Incontinence Treatment Network studies were analyzed. White women comprised 70%-89% of PFD literature; Black women, 6%-16%; Hispanic women, 9%-15%; Asians, 0.5%-6%; and American Indians, 0%-2%. Representation of White women was higher in 13 of 18 PFDN studies compared with the NHANES prevalence data. Representation of Black women was either decreased or not reported in 10 of 18 index studies compared with the NHANES prevalence data. Hispanic women were absent or underrepresented in 7 of 18 PFDN studies compared with the prevalence data. CONCLUSIONS: Our examination of PFDN and other landmark trials demonstrates inconsistent reporting of minority subgroups, limiting applicability with respect to minority populations. Our study suggests that PFD research would benefit from targeted sampling of minority groups.

The West coast regional safety pharmacology society meeting update: Filling translational gaps in safety assessment.

The Safety Pharmacology Society (SPS) held a West Coast Regional Meeting in Foster City, CA on November 14, 2018 at the Gilead Sciences Inc. site. The meeting was attended by scientists from the pharmaceutical and biotechnology industry, contract research organizations (CROs) and academia. A variety of scientific topics were presented by speakers, covering a broad variety of topics in the fields of safety risk assessment; from pro-arrhythmia and contractility risk evaluation, to models of heart failure and seizure in-a-dish; and discovery sciences; from stem cells and precision medicine, to models of inherited cardiomyopathy and precision cut tissue slices. The present review summarizes the highlights of the presentations and provides an overview of the high level of innovation currently underlying many frontiers in safety pharmacology.

Changes observed in prostate biopsy practices in an inner city hospital with a high risk patient population following the 2012 USPSTF PSA screening recommendations

ABSTRACT Introduction: We compared characteristics of patients undergoing prostate biopsy in a high-risk inner city population before and after the 2012 USPSTF recommendation against PSA based prostate cancer screening to determine its effect on prostate biopsy practices. Materials and Methods: This was a retrospective study including patients who received biopsies after an abnormal PSA measurement from October 2008-December 2015. Patients with previously diagnosed prostate cancer were excluded. Chi-square tests of independence, two sample t-tests, Mann-Whitney U tests, and Fisher's exact tests were performed. Results: There were 202 and 208 patients in the pre-USPSTF and post-USPSTF recommendation cohorts, respectively. The post-USPSTF cohort had higher median PSA (7.8 versus 7.1ng/mL, p=0.05), greater proportion of patients who were black (96.6% versus 90.5%, p=0.01), and greater percentage of biopsy cores positive for disease (58% versus 29.5%, p<0.001). Multivariable analysis supported that the increase in PSA was independent of the increase in the proportion of patients who were black. The proportion of patients who were classified as D'Amico intermediate and high-risk disease increased in the post-USPSTF cohort and approached statistical significance (70.1% versus 58.8%, p=0.12). Conclusions: Our study suggests that the USPSTF recommendations may have led to an increase in pre-biopsy PSA as well as greater volume of disease. Also, a greater proportion of patients were being classified with intermediate or high risk disease. While the clinical significance of these findings is unknown, what the data suggests is somewhat troubling. Future research should further examine these changes in a larger cohort as well as resultant long-term outcomes.

Changes observed in prostate biopsy practices in an inner city hospital with a high risk patient population following the 2012 uspstf psa screening recommendations.

INTRODUCTION: We compared characteristics of patients undergoing prostate biopsy in a high-risk inner city population before and after the 2012 USPSTF recommendation against PSA based prostate cancer screening to determine its effect on prostate biopsy practices. MATERIALS AND METHODS: This was a retrospective study including patients who received biopsies after an abnormal PSA measurement from October 2008-December 2015. Patients with previously diagnosed prostate cancer were excluded. Chi-square tests of independence, two sample t-tests, Mann-Whitney U tests, and Fisher's exact tests were performed. RESULTS: There were 202 and 208 patients in the pre-USPSTF and post-USPSTF recommendation cohorts, respectively. The post-USPSTF cohort had higher median PSA (7.8 versus 7.1ng/mL, p=0.05), greater proportion of patients who were black (96.6% versus 90.5%, p=0.01), and greater percentage of biopsy cores positive for disease (58% versus 29.5%, p<0.001). Multivariable analysis supported that the increase in PSA was independent of the increase in the proportion of patients who were black. The proportion of patients who were classified as D'Amico intermediate and high-risk disease increased in the post-USPSTF cohort and approached statistical significance (70.1% versus 58.8%, p=0.12). CONCLUSIONS: Our study suggests that the USPSTF recommendations may have led to na increase in pre-biopsy PSA as well as greater volume of disease. Also, a greater proportion of patients were being classified with intermediate or high risk disease. While the clinical significance of these findings is unknown, what the data suggests is somewhat troubling. Future research should further examine these changes in a larger cohort as well as resultant long-term outcomes.

Surgical treatment of detrusor underactivity: a short term proof of concept study

ABSTRACT Objectives To compare the surgical outcomes of men with bladder outlet obstruction (BOO) due to benign prostatic obstruction (BPO) to those with detrusor underactivity (DU) or acontractile detrusor (DA). Materials and Methods This retrospective, IRB approved study included men who underwent BPO surgery for refractory LUTS or urinary retention. Patients were grouped based on videourodynamic (VUDS) findings: 1) men with BOO, 2) men with DU and 3) men with DA. The primary outcome measure was the Patient Global Impression of Improvement (PGII). Secondary outcome measures included uroflow (Qmax), post-void residual volume (PVR) and the need for clean intermittent catheterization (CIC). Results One hundred and nineteen patients were evaluated: 1) 34 with BOO, 2) 62 with DU and 3) 23 with DA. Subjective success rate (PGII) was highest in the BOO group (97%) and those with DU (98%), while DA patients had a PGII success of 26%, (p<0.0001). After surgery, patients with BOO had the lowest PVR (68.5mL). Fifty-six patients (47%) performed CIC pre-operatively (47% of BOO, 32% of DU and 87% of DA patients). None of the patients in the BOO and DU groups required CIC post operatively compared to16/23 (69%) of patients in the DA group (p<0.0001). Conclusions BPO surgery is a viable treatment option in men with presumed BOO and DU while DA is a poor prognostic sign in men who do not void spontaneously pre-operatively.

Urogynecological conditions associated with overactive bladder symptoms in women.

INTRODUCTION: Overactive bladder symptoms (OAB) affect 9-43% of women and are associated with underlying disorders, including pelvic organ prolapse (POP) and stress urinary incontinence (SUI). The aim of this study is to identify urogynecological conditions associated with OAB symptoms. METHODS: This prospective, institutional review board-approved study included women referred to a tertiary centre with lower urinary tract symptoms (LUTS). All women completed the self-administered OAB questionnaire (OABSS). Those with an OABSS ≥8, the cutoff, were considered to have OAB symptoms. Patients underwent a history and physical examination (including Baden-Walker prolapse grading and stress test), 24-hour voiding diary, pad test (for urinary incontinence), urinalysis, and uroflow with post-void residual volume. Patients were classified clinically into the following: idiopathic OAB, SUI, POP, bladder outlet obstruction (BOO) neurogenic bladder (NGB), recurrent urinary tract infection (UTI), and miscellaneous. RESULTS: In total, 148 women met the inclusion criteria with a mean age of 67 years. Only 27% had no comorbid conditions and were considered idiopathic OAB. Associated urogynecological conditions included SUI in 37%, POP in 26%, miscellaneous conditions in 18%, recurrent UTI in 11%, NGB in 9%, and BOO in 8%. Some patients met criteria for more than one category, thus the total is greater than 100%. CONCLUSIONS: In a tertiary care setting, a significant proportion of women with OAB symptoms have underlying conditions that may cause or contribute to their symptoms. Appropriate evaluation is desirable to enhance our understanding of the relationship of these conditions to the diagnosis, treatment, outcomes, and pathophysiology of OAB.

Surgical treatment of detrusor underactivity: a short term proof of concept study.

OBJECTIVES: To compare the surgical outcomes of men with bladder outlet obstruction (BOO) due to benign prostatic obstruction (BPO) to those with detrusor underactivity (DU) or acontractile detrusor (DA). MATERIALS AND METHODS: This retrospective, IRB approved study included men who underwent BPO surgery for refractory LUTS or urinary retention. Patients were grouped based on videourodynamic (VUDS) findings: 1) men with BOO, 2) men with DU and 3) men with DA. The primary outcome measure was the Patient Global Impression of Improvement (PGII). Secondary outcome measures included uroflow (Qmax), post-void residual volume (PVR) and the need for clean intermittent catheterization (CIC). RESULTS: One hundred and nineteen patients were evaluated: 1) 34 with BOO, 2) 62 with DU and 3) 23 with DA. Subjective success rate (PGII) was highest in the BOO group (97%) and those with DU (98%), while DA patients had a PGII success of 26%, (p<0.0001). After surgery, patients with BOO had the lowest PVR (68.5mL). Fifty-six patients (47%) performed CIC pre-operatively (47% of BOO, 32% of DU and 87% of DA patients). None of the patients in the BOO and DU groups required CIC post operatively compared to16/23 (69%) of patients in the DA group (p<0.0001). CONCLUSIONS: BPO surgery is a viable treatment option in men with presumed BOO and DU while DA is a poor prognostic sign in men who do not void spontaneously pre-operatively.

The primate-specific noncoding RNA HPAT5 regulates pluripotency during human preimplantation development and nuclear reprogramming.

Long intergenic noncoding RNAs (lincRNAs) are derived from thousands of loci in mammalian genomes and are frequently enriched in transposable elements (TEs). Although families of TE-derived lincRNAs have recently been implicated in the regulation of pluripotency, little is known of the specific functions of individual family members. Here we characterize three new individual TE-derived human lincRNAs, human pluripotency-associated transcripts 2, 3 and 5 (HPAT2, HPAT3 and HPAT5). Loss-of-function experiments indicate that HPAT2, HPAT3 and HPAT5 function in preimplantation embryo development to modulate the acquisition of pluripotency and the formation of the inner cell mass. CRISPR-mediated disruption of the genes for these lincRNAs in pluripotent stem cells, followed by whole-transcriptome analysis, identifies HPAT5 as a key component of the pluripotency network. Protein binding and reporter-based assays further demonstrate that HPAT5 interacts with the let-7 microRNA family. Our results indicate that unique individual members of large primate-specific lincRNA families modulate gene expression during development and differentiation to reinforce cell fate.

Minimally Invasive Valve Surgery and Single Vessel Coronary Artery Bypass via Limited Anterior Right Thoracotomy.

BACKGROUND: Coronary artery bypass grafting with aortic valve replacement (AVR) or mitral valve replacement (MVR) is traditionally performed via sternotomy. Minimally invasive coronary surgery (MICS) and minimally invasive valve surgery have been successfully performed independently. Patients with critical right coronary artery (RCA) stenosis not amenable to percutaneous intervention are candidates for valve replacement and single vessel coronary artery bypass. We present our series of six patients who underwent a concomitant valve and single vessel intervention via right thoracotomy. METHODS: Between January 2011 and June 2013, six patients underwent right thoracotomy with valve replacement and single vessel bypass. Four aortic and two mitral valves were replaced and all received single vessel RCA bypass using reversed saphenous vein graft. Thoracotomy was via right anterior approach for AVR and right lateral for MVR. The patients were assessed postoperatively for overall outcomes. RESULTS: The average age was 74 years (range 69-81); two patients were elective (AVR-1; MVR-1) and four were urgent (AVR-3; MVR-1). For MICS AVR and MICS MVR, the average cardiopulmonary bypass time was 171 ± 30 and 169 ± 7 minutes and the average aortic cross-clamp time was 122 ± 36 and 112 ± 2 minutes, respectively. Three patients were discharged home, one patient to a nursing home, and two to rehab. No patients required conversion to sternotomy; one patient developed atrial fibrillation, and one sepsis. CONCLUSION: Concomitant valve replacement and single bypass grafting via right anterior mini-thoracotomy is a viable option for select patients, particularly in non-stentable RCA stenosis. In the appropriate patient population, combined coronary artery bypass grafting and valve surgery can be safely performed via right thoracotomy.

Generation of induced neuronal cells by the single reprogramming factor ASCL1.

Direct conversion of nonneural cells to functional neurons holds great promise for neurological disease modeling and regenerative medicine. We previously reported rapid reprogramming of mouse embryonic fibroblasts (MEFs) into mature induced neuronal (iN) cells by forced expression of three transcription factors: ASCL1, MYT1L, and BRN2. Here, we show that ASCL1 alone is sufficient to generate functional iN cells from mouse and human fibroblasts and embryonic stem cells, indicating that ASCL1 is the key driver of iN cell reprogramming in different cell contexts and that the role of MYT1L and BRN2 is primarily to enhance the neuronal maturation process. ASCL1-induced single-factor neurons (1F-iN) expressed mature neuronal markers, exhibited typical passive and active intrinsic membrane properties, and formed functional pre- and postsynaptic structures. Surprisingly, ASCL1-induced iN cells were predominantly excitatory, demonstrating that ASCL1 is permissive but alone not deterministic for the inhibitory neuronal lineage.

Calcineurin signaling regulates neural induction through antagonizing the BMP pathway.

Development of the nervous system begins with neural induction, which is controlled by complex signaling networks functioning in concert with one another. Fine-tuning of the bone morphogenetic protein (BMP) pathway is essential for neural induction in the developing embryo. However, the molecular mechanisms by which cells integrate the signaling pathways that contribute to neural induction have remained unclear. We find that neural induction is dependent on the Ca(2+)-activated phosphatase calcineurin (CaN). Fibroblast growth factor (FGF)-regulated Ca(2+) entry activates CaN, which directly and specifically dephosphorylates BMP-regulated Smad1/5 proteins. Genetic and biochemical analyses revealed that CaN adjusts the strength and transcriptional output of BMP signaling and that a reduction of CaN activity leads to an increase of Smad1/5-regulated transcription. As a result, FGF-activated CaN signaling opposes BMP signaling during gastrulation, thereby promoting neural induction and the development of anterior structures.

A positive feedback mechanism that regulates expression of miR-9 during neurogenesis.

MiR-9, a neuron-specific miRNA, is an important regulator of neurogenesis. In this study we identify how miR-9 is regulated during early differentiation from a neural stem-like cell. We utilized two immortalized rat precursor clones, one committed to neurogenesis (L2.2) and another capable of producing both neurons and non-neuronal cells (L2.3), to reproducibly study early neurogenesis. Exogenous miR-9 is capable of increasing neurogenesis from L2.3 cells. Only one of three genomic loci capable of encoding miR-9 was regulated during neurogenesis and the promoter region of this locus contains sufficient functional elements to drive expression of a luciferase reporter in a developmentally regulated pattern. Furthermore, among a large number of potential regulatory sites encoded in this sequence, Mef2 stood out because of its known pro-neuronal role. Of four Mef2 paralogs, we found only Mef2C mRNA was regulated during neurogenesis. Removal of predicted Mef2 binding sites or knockdown of Mef2C expression reduced miR-9-2 promoter activity. Finally, the mRNA encoding the Mef2C binding partner HDAC4 was shown to be targeted by miR-9. Since HDAC4 protein could be co-immunoprecipitated with Mef2C protein or with genomic Mef2 binding sequences, we conclude that miR-9 regulation is mediated, at least in part, by Mef2C binding but that expressed miR-9 has the capacity to reduce inhibitory HDAC4, stabilizing its own expression in a positive feedback mechanism.

Acute reduction in oxygen tension enhances the induction of neurons from human fibroblasts.

We and others have reported the successful conversion of human fibroblasts into functional induced neuronal (iN) cells; however the reprogramming efficiencies were very low. Robust reprogramming methods must be developed before iN cells can be used for translational applications such as disease modeling or transplantation-based therapies. Here, we describe a novel approach in which we significantly enhance iN cell conversion efficiency of human fibroblast cells by reprogramming under hypoxic conditions (5% O2). Fibroblasts were derived under high (21%) or low (5%) oxygen conditions and reprogrammed into iN cells using a combination of the four transcription factors BRN2, ASCL1, MYT1L and NEUROD1. An increase in Map2 immunostaining was only observed when fibroblasts experienced an acute drop in O2 tension upon infection. Interestingly, cells derived and reprogrammed under hypoxic conditions did not produce more iN cells. Approximately 100% of patched cells fired action potentials in low O2 conditions compared to 50% under high O2 growth conditions, confirming the beneficial aspect of reprogramming under low O2. Further characterization showed no significant difference in the intrinsic properties of iN cells reprogrammed in either condition. Surprisingly, the acute drop in oxygen tension did not affect cell proliferation or cell survival and was not synergistic with the blockade of GSK3ß and Smad-mediated pathways. Our results showed that lowering the O2 tension at the initiation of reprogramming is a simple and efficient strategy to enhance the production of iN cells which will facilitate their use for basic discovery and regenerative medicine.

Expression profiling of synaptic microRNAs from the adult rat brain identifies regional differences and seizure-induced dynamic modulation.

In recent years, microRNAs or miRNAs have been proposed to target neuronal mRNAs localized near the synapse, exerting a pivotal role in modulating local protein synthesis, and presumably affecting adaptive mechanisms such as synaptic plasticity. In the present study we have characterized the distribution of miRNAs in five regions of the adult mammalian brain and compared the relative abundance between total fractions and purified synaptoneurosomes (SN), using three different methodologies. The results show selective enrichment or depletion of some miRNAs when comparing total versus SN fractions. These miRNAs were different for each brain region explored. Changes in distribution could not be attributed to simple diffusion or to a targeting sequence inside the miRNAs. In silico analysis suggest that the differences in distribution may be related to the preferential concentration of synaptically localized mRNA targeted by the miRNAs. These results favor a model of co-transport of the miRNA-mRNA complex to the synapse, although further studies are required to validate this hypothesis. Using an in vivo model for increasing excitatory activity in the cortex and the hippocampus indicates that the distribution of some miRNAs can be modulated by enhanced neuronal (epileptogenic) activity. All these results demonstrate the dynamic modulation in the local distribution of miRNAs from the adult brain, which may play key roles in controlling localized protein synthesis at the synapse.

MicroRNA miR-133b is essential for functional recovery after spinal cord injury in adult zebrafish.

MicroRNAs (miRNAs) play important roles during development and also in adult organisms by regulating the expression of multiple target genes. Here, we studied the function of miR-133b during zebrafish spinal cord regeneration and show upregulation of miR-133b expression in regenerating neurons of the brainstem after transection of the spinal cord. miR-133b has been shown to promote tissue regeneration in other tissue, but its ability to do so in the nervous system has yet to be tested. Inhibition of miR-133b expression by antisense morpholino (MO) application resulted in impaired locomotor recovery and reduced regeneration of axons from neurons in the nucleus of the medial longitudinal fascicle, superior reticular formation and intermediate reticular formation. miR-133b targets the small GTPase RhoA, which is an inhibitor of axonal growth, as well as other neurite outgrowth-related molecules. Our results indicate that miR-133b is an important determinant in spinal cord regeneration of adult zebrafish through reduction in RhoA protein levels by direct interaction with its mRNA. While RhoA has been studied as a therapeutic target in spinal cord injury, this is the first demonstration of endogenous regulation of RhoA by a microRNA that is required for spinal cord regeneration in zebrafish. The ability of miR-133b to suppress molecules that inhibit axon regrowth may underlie the capacity for adult zebrafish to recover locomotor function after spinal cord injury.