Author Correction: Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Super-Mendelian inheritance mediated by CRISPR-Cas9 in the female mouse germline.

A gene drive biases the transmission of one of the two copies of a gene such that it is inherited more frequently than by random segregation. Highly efficient gene drive systems have recently been developed in insects, which leverage the sequence-targeted DNA cleavage activity of CRISPR-Cas9 and endogenous homology-directed repair mechanisms to convert heterozygous genotypes to homozygosity1-4. If implemented in laboratory rodents, similar systems would enable the rapid assembly of currently impractical genotypes that involve multiple homozygous genes (for example, to model multigenic human diseases). To our knowledge, however, such a system has not yet been demonstrated in mammals. Here we use an active genetic element that encodes a guide RNA, which is embedded in the mouse tyrosinase (Tyr) gene, to evaluate whether targeted gene conversion can occur when CRISPR-Cas9 is active in the early embryo or in the developing germline. Although Cas9 efficiently induces double-stranded DNA breaks in the early embryo and male germline, these breaks are not corrected by homology-directed repair. By contrast, Cas9 expression limited to the female germline induces double-stranded breaks that are corrected by homology-directed repair, which copies the active genetic element from the donor to the receiver chromosome and increases its rate of inheritance in the next generation. These results demonstrate the feasibility of CRISPR-Cas9-mediated systems that bias inheritance of desired alleles in mice and that have the potential to transform the use of rodent models in basic and biomedical research.

Gene expression differences associated with intrinsic hindfoot muscle loss in the jerboa, Jaculus jaculus.

Vertebrate animals that run or jump across sparsely vegetated habitats, such as horses and jerboas, have reduced the number of distal limb bones, and many have lost most or all distal limb muscle. We previously showed that nascent muscles are present in the jerboa hindfoot at birth and that these myofibers are rapidly and completely lost soon after by a process that shares features with pathological skeletal muscle atrophy. Here, we apply an intra- and interspecies differential RNA-Seq approach, comparing jerboa and mouse muscles, to identify gene expression differences associated with the initiation and progression of jerboa hindfoot muscle loss. We show evidence for reduced hepatocyte growth factor and fibroblast growth factor signaling and an imbalance in nitric oxide signaling; all are pathways that are necessary for skeletal muscle development and regeneration. We also find evidence for phagosome formation, which hints at how myofibers may be removed by autophagy or by nonprofessional phagocytes without evidence for cell death or immune cell activation. Last, we show significant overlap between genes associated with jerboa hindfoot muscle loss and genes that are differentially expressed in a variety of human muscle pathologies and rodent models of muscle loss disorders. All together, these data provide molecular insight into the process of evolutionary and developmental muscle loss in jerboa hindfeet.

Meiotic Cas9 expression mediates gene conversion in the male and female mouse germline.

Highly efficient gene conversion systems have the potential to facilitate the study of complex genetic traits using laboratory mice and, if implemented as a "gene drive," to limit loss of biodiversity and disease transmission caused by wild rodent populations. We previously showed that such a system of gene conversion from heterozygous to homozygous after a sequence targeted CRISPR/Cas9 double-strand DNA break (DSB) is feasible in the female mouse germline. In the male germline, however, all DSBs were instead repaired by end joining (EJ) mechanisms to form an "insertion/deletion" (indel) mutation. These observations suggested that timing Cas9 expression to coincide with meiosis I is critical to favor conditions when homologous chromosomes are aligned and interchromosomal homology-directed repair (HDR) mechanisms predominate. Here, using a Cas9 knock-in allele at the Spo11 locus, we show that meiotic expression of Cas9 does indeed mediate gene conversion in the male as well as in the female germline. However, the low frequency of both HDR and indel mutation in both male and female germlines suggests that Cas9 may be expressed from the Spo11 locus at levels too low for efficient DSB formation. We suggest that more robust Cas9 expression initiated during early meiosis I may improve the efficiency of gene conversion and further increase the rate of "super-mendelian" inheritance from both male and female mice.

Assessment of health-related quality of life and health status in patients with treatment-resistant depression treated with esketamine nasal spray plus an oral antidepressant.

BACKGROUND: Patients with treatment-resistant depression (TRD) report significant deficits in physical and mental health, as well as severely impaired health-related quality of life (HRQoL) and functioning. Esketamine effectively enhances the daily functioning in these patients while also improving their depressive symptoms. This study assessed HRQoL and health status of patients with TRD, who were treated with esketamine nasal spray and an oral antidepressant (ESK + AD) vs. placebo nasal spray and an AD (AD + PBO). METHODS: Data from TRANSFORM-2, a phase 3, randomized, double-blind, short-term flexibly dosed study, were analyzed. Patients (aged 18-64 years) with TRD were included. The outcome assessments included the European Quality of Life Group, Five Dimension, Five Level (EQ-5D-5L), EQ-Visual Analogue Scale (EQ-VAS), and Sheehan Disability Scale (SDS). The health status index (HSI) was calculated using EQ-5D-5L scores. RESULTS: The full analysis set included 223 patients (ESK + AD: 114; AD + PBO: 109; mean [SD] age: 45.7 [11.89]). At Day 28, a lower percentage of patients reported impairment in the ESK + AD vs. AD + PBO group in all five EQ-5D-5L dimensions: mobility (10.6% vs. 25.0%), self-care (13.5% vs. 32.0%), usual activities (51.9% vs. 72.0%), pain/discomfort (35.6% vs. 54.0%), and anxiety/depression (69.2% vs. 78.0%). The mean (SD) change from baseline in HSI at Day 28 was 0.310 (0.219) for ESK + AD and 0.235 (0.252) for AD + PBO, with a higher score reflecting better levels of health. The mean (SD) change from baseline in EQ-VAS score at Day 28 was greater in ESK + AD (31.1 [25.67]) vs. AD + PBO (22.1 [26.43]). The mean (SD) change in the SDS total score from baseline to Day 28 also favored ESK + AD (-13.6 [8.31]) vs. AD + PBO (-9.4 [8.43]). CONCLUSIONS: Greater improvements in HRQoL and health status were observed among patients with TRD treated with ESK + AD vs. AD + PBO. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02418585.

Metatarsal fusion resisted bending as jerboas (Dipodidae) transitioned from quadrupedal to bipedal.

Hind limbs undergo dramatic changes in loading conditions during the transition from quadrupedal to bipedal locomotion. For example, the most early diverging bipedal jerboas (Rodentia: Dipodidae) are some of the smallest mammals in the world, with body masses that range between 2-4 g. The larger jerboa species exhibit developmental and evolutionary fusion of the central three metatarsals into a single cannon bone. We hypothesize that small body size and metatarsal fusion are mechanisms to maintain the safety factor of the hind limb bones despite the higher ground reaction forces associated with bipedal locomotion. Using finite-element analysis to model collisions between the substrate and the metatarsals, we found that body size reduction was insufficient to reduce bone stress on unfused metatarsals, based on the scaled dynamics of larger jerboas, and that fused bones developed lower stresses than unfused bones when all metatarsals are scaled to the same size and loading conditions. Based on these results, we conclude that fusion reinforces larger jerboa metatarsals against high ground reaction forces. Because smaller jerboas with unfused metatarsals develop higher peak stresses in response to loading conditions scaled from larger jerboas, we hypothesize that smaller jerboas use alternative dynamics of bipedal locomotion to reduce the impact of collisions between the foot and substrate.

Efficacy and safety of esketamine nasal spray by sex in patients with treatment-resistant depression: findings from short-term randomized, controlled trials.

The objective of this analysis was to determine if there are sex differences with esketamine for treatment-resistant depression (TRD). Post hoc analyses of three randomized, controlled studies of esketamine in patients with TRD (TRANSFORM-1, TRANSFORM-2 [18-64 years], TRANSFORM-3 [≥ 65 years]) were performed. In each 4-week study, adults with TRD were randomized to esketamine or placebo nasal spray, each with a newly initiated oral antidepressant. Change from baseline to day 28 in Montgomery-Åsberg Depression Rating Scale (MADRS) total score was assessed by sex in pooled data from TRANSFORM-1/TRANSFORM-2 and separately in data from TRANSFORM-3 using a mixed-effects model for repeated measures. Use of hormonal therapy was assessed in all women, and menopausal status was assessed in women in TRANSFORM-1/TRANSFORM-2. Altogether, 702 adults (464 women) received ≥ 1 dose of intranasal study drug and antidepressant. Mean MADRS total score (SD) decreased from baseline to day 28, more so among patients treated with esketamine/antidepressant vs. antidepressant/placebo in both women and men: TRANSFORM-1/TRANSFORM-2 women-esketamine/antidepressant -20.3 (13.19) vs. antidepressant/placebo -15.8 (14.67), men-esketamine/antidepressant -18.3 (14.08) vs. antidepressant/placebo -16.0 (14.30); TRANSFORM-3 women-esketamine/antidepressant -9.9 (13.34) vs. antidepressant/placebo -6.9 (9.65), men-esketamine/antidepressant -10.3 (11.96) vs. antidepressant/placebo -5.5 (7.64). There was no significant sex effect or treatment-by-sex interaction (p > 0.35). The most common adverse events in esketamine-treated patients were nausea, dissociation, dizziness, and vertigo, each reported at a rate higher in women than men. The analyses support antidepressant efficacy and overall safety of esketamine nasal spray are similar between women and men with TRD. The TRANSFORM studies are registered at clinicaltrials.gov (identifiers: NCT02417064 (first posted 15 April 2015; last updated 4 May 2020), NCT02418585 (first posted 16 April 2015; last updated 2 June 2020), and NCT02422186 (first posted 21 April 2015; last updated 29 September 2021)).

Inhibition of dual-specificity tyrosine phosphorylation-regulated kinase 2 perturbs 26S proteasome-addicted neoplastic progression.

Dependence on the 26S proteasome is an Achilles' heel for triple-negative breast cancer (TNBC) and multiple myeloma (MM). The therapeutic proteasome inhibitor, bortezomib, successfully targets MM but often leads to drug-resistant disease relapse and fails in breast cancer. Here we show that a 26S proteasome-regulating kinase, DYRK2, is a therapeutic target for both MM and TNBC. Genome editing or small-molecule mediated inhibition of DYRK2 significantly reduces 26S proteasome activity, bypasses bortezomib resistance, and dramatically delays in vivo tumor growth in MM and TNBC thereby promoting survival. We further characterized the ability of LDN192960, a potent and selective DYRK2-inhibitor, to alleviate tumor burden in vivo. The drug docks into the active site of DYRK2 and partially inhibits all 3 core peptidase activities of the proteasome. Our results suggest that targeting 26S proteasome regulators will pave the way for therapeutic strategies in MM and TNBC.

Reducing overprescribing of antibiotics for suspected urinary tract infections in a health sciences campus student health service.

AIMS: Recommendations for the management of women with suspected uncomplicated lower urinary tract infections (UTIs) include presumptive antibiotics with or without obtaining a urine culture (UCx). However, with increasing antibiotic resistance, efforts to decrease antibiotic usage are vital. Therefore, the objective of this study was to determine if the presumptive treatment of women with suspected uncomplicated UTIs is contributing to unnecessary antibiotic usage. METHODS: We retrospectively reviewed all nonpregnant female patients presenting to our student health services clinic with UTI symptoms from December 2016 to May 2017 who had UCx sent. Clinical information, symptoms, office urine dip, and UCx results were reviewed. Patients with positive and negative UCx were compared. RESULTS: A total of 67 patients were included for analysis. Presenting symptoms included dysuria (59/60, 98%), frequency (41/45, 91%), and urgency (27/27, 100%). Office urine dip was performed on 33 of 67 (49%) patients. Dips were positive for leukocytes (88%), blood (79%), and nitrites (18%). All patients in the study were prescribed antibiotics, most commonly nitrofurantoin (82%). Culture results were negative in 29 of 67 (43%). There were no significant differences in duration of symptoms, presenting symptoms, or urine dip results between patients with a negative UCx and those with a positive UCx. CONCLUSIONS: In our study, we found a significant negative UCx rate in women with symptoms of uncomplicated UTI, representing a cohort of patients who were exposed to antibiotics unnecessarily. In addition, we found no difference in presenting symptoms or urine dip results to help distinguish patients with a positive UCx.

Patterning and post-patterning modes of evolutionary digit loss in mammals.

A reduction in the number of digits has evolved many times in tetrapods, particularly in cursorial mammals that travel over deserts and plains, yet the underlying developmental mechanisms have remained elusive. Here we show that digit loss can occur both during early limb patterning and at later post-patterning stages of chondrogenesis. In the 'odd-toed' jerboa (Dipus sagitta) and horse and the 'even-toed' camel, extensive cell death sculpts the tissue around the remaining toes. In contrast, digit loss in the pig is orchestrated by earlier limb patterning mechanisms including downregulation of Ptch1 expression but no increase in cell death. Together these data demonstrate remarkable plasticity in the mechanisms of vertebrate limb evolution and shed light on the complexity of morphological convergence, particularly within the artiodactyl lineage.

Convergent metatarsal fusion in jerboas and chickens is mediated by similarities and differences in the patterns of osteoblast and osteoclast activities.

In many vertebrate animals that run or leap, the metatarsals and/or metacarpals of the distal limb are fused into a single larger element, likely to resist fracture due to high ground-reaction forces during locomotion. Although metapodial fusion evolved independently in modern birds, ungulates, and jerboas, the developmental basis has only been explored in chickens, which diverged from the mammalian lineage approximately 300 million years ago. Here, we use a bipedal rodent, the lesser Egyptian jerboa (Jaculus jaculus), to understand the cellular processes of metatarsal fusion in a mammal, and we revisit the developing chicken to assess similarities and differences in the localization of osteoblast and osteoclast activities. In both species, adjacent metatarsals align along flat surfaces, osteoblasts cross the periosteal membrane to unite the three elements in a single circumference, and osteoclasts resorb bone at the interfaces leaving a single marrow cavity. However, the pattern of osteoclast activity differs in each species; osteoclasts are highly localized to resorb bone at the interfaces of neighboring jerboa metatarsals and are distributed throughout the endosteum of chicken metatarsals. Each species, therefore, provides an opportunity to understand mechanisms that pattern osteoblast and osteoclast activities to alter bone shape during development and evolution.

Multiple phases of chondrocyte enlargement underlie differences in skeletal proportions.

The wide diversity of skeletal proportions in mammals is evident upon a survey of any natural history museum's collections and allows us to distinguish between species even when reduced to their calcified components. Similarly, each individual is comprised of a variety of bones of differing lengths. The largest contribution to the lengthening of a skeletal element, and to the differential elongation of elements, comes from a dramatic increase in the volume of hypertrophic chondrocytes in the growth plate as they undergo terminal differentiation. However, the mechanisms of chondrocyte volume enlargement have remained a mystery. Here we use quantitative phase microscopy to show that mammalian chondrocytes undergo three distinct phases of volume increase, including a phase of massive cell swelling in which the cellular dry mass is significantly diluted. In light of the tight fluid regulatory mechanisms known to control volume in many cell types, this is a remarkable mechanism for increasing cell size and regulating growth rate. It is, however, the duration of the final phase of volume enlargement by proportional dry mass increase at low density that varies most between rapidly and slowly elongating growth plates. Moreover, we find that this third phase is locally regulated through a mechanism dependent on insulin-like growth factor. This study provides a framework for understanding how skeletal size is regulated and for exploring how cells sense, modify and establish a volume set point.

Utility of catheterized specimens in reducing overdiagnosis of urinary tract infections in women.

AIMS: To evaluate the utility of catheterized samples in reducing overdiagnosis of UTI based on voided specimens among patients presenting with a range of urinary symptoms. We also aimed to determine variables that may modify the predictive value of the voided midstream urine culture. METHODS: Patient charts were reviewed to identify female patients referred to our voiding dysfunction clinic with a range of complaints warranting urine studies (5/2014-8/2016). Patients with a positive voided urine culture who also had a catheterized urine culture in our system were included. Multiple logistic regression analysis was performed to identify patient characteristics associated with a negative catheterized specimen despite a positive voided specimen. RESULTS: One hundred and seven women were included in the study. Eighty percent of the cohort was post-menopausal. Although all patients had positive voided specimens, only 53 (49.5%) had positive catheterized specimens. On multivariate analysis negative nitrites on clean catch UA was a significant predictor of a negative catheterized sample (adjusted OR 8.9, 95%CI 2.2-43.7, P = 0.003). WBC/HPF <10 on clean catch UA trended towards significance (adjusted OR 4.72, 95%CI 1.1-26.1, P = 0.05). CONCLUSIONS: Relying on clean catch urine samples may lead to significant over-diagnosis of UTIs. Our study suggests that in female patients who have vague symptoms of UTI, obtaining catheterized specimens may be beneficial in avoiding the overdiagnosis of UTIs and the overuse of antibiotics. Larger, prospective studies testing our hypothesis are necessary, and would greatly assist in establishing clinical practices that reduce the amount of antibiotics inappropriately prescribed.

Assessment of practices in screening and treating women with bacteriuria.

INTRODUCTION: Evidence-based screening and treatment for bacteriuria is crucial to prevent increasing antibiotic resistance. The Infectious Disease Society of America (IDSA) previously released guidelines on the management of asymptomatic bacteriuria (ASB) and uncomplicated urinary tract infections (UTIs) in women. The study's objective was to assess physicians' practices in managing women with bacteriuria relative to these guideline recommendations. MATERIALS AND METHODS: Cross-sectional data from physicians were collected using an anonymous questionnaire. Multivariable logistic regression analyses identified independent predictors of adherence to guidelines. RESULTS: Data were collected from 260 physicians. Over half of physicians surveyed were unfamiliar with IDSA guidelines and overtreat ASB. Variables independently associated with overtreatment of ASB included a non-academic practice and practicing as an OBGYN. Nearly one third (30.1%) of physicians reported prescribing an antibiotic other than a recommended first-line agent for uncomplicated cystitis. Relative to internists, OBGYNs and urologists were more likely to prescribe a recommended first-line agent to women with uncomplicated cystitis. Of those who correctly selected a first-line agent, 29.8% prescribed a longer than recommended duration of therapy. IDSA guideline awareness was not associated with physicians' practices in managing women with bacteriuria. CONCLUSIONS: Most physicians surveyed were unfamiliar with guidelines related to managing ASB and uncomplicated UTIs in women, likely contributing to overscreening and overtreatment of ASB and the use of inappropriate antibiotic regimens in treating uncomplicated cystitis. However, optimal antibiotic prescribing was not associated with knowledge of IDSA guidelines, suggesting that guideline dissemination alone may not alter practice patterns among physicians managing women with bacteriuria.

Diagnostic yield of cystoscopy in the evaluation of recurrent urinary tract infection in women.

AIMS: Due to a paucity of evidence-based guidelines, anecdotal practice patterns often dictate clinical management of recurrent urinary tract infection (UTI) in women. Our aim was to identify pathologic findings of the urinary tract through cystoscopy and imaging in women with recurrent UTI, and to determine if specific risk factors are associated with a higher rate of abnormal findings. METHODS: In a single-institutional cohort, cystoscopy was performed for women with recurrent UTI between 1/2010 and 7/2014. All eligible patients were included in a maintained database and those with gross or microscopic hematuria were excluded. Abdominopelvic imaging was recommended and included in study data when completed. Associations between clinical risk factors (history of renal transplant, urogynecologic surgery, or urolithiasis) and abnormal findings were analyzed by Fisher's exact test. RESULTS: A total of 163 women (mean age 60.6 years) were included in final analysis. Abdominopelvic imaging was available in 133 (82%) cases. Cystoscopy identified 9 (5.5%) cases of significant clinical findings. Of these only 5 (3.8%) cases were uniquely identified on cystoscopy and missed on imaging modalities. When imaging was normal, cystoscopy was also normal in 94% of cases. The examined clinical risk factors were not associated with higher risk of abnormal cystoscopy (P = 0.49) or imaging (P = 0.42). CONCLUSIONS: Cystoscopy performed solely for recurrent UTI is low yield in patients with normal imaging studies, but a small number of abnormal findings may be missed by foregoing this element of the patient workup. No studied risk factor was predictive of an abnormal workup. Neurourol. Urodynam. 36:692-696, 2017. © 2016 Wiley Periodicals, Inc.

Unique expression patterns of multiple key genes associated with the evolution of mammalian flight.

Bats are the only mammals capable of true flight. Critical adaptations for flight include a pair of dramatically elongated hands with broad wing membranes. To study the molecular mechanisms of bat wing evolution, we perform genomewide mRNA sequencing and in situ hybridization for embryonic bat limbs. We identify seven key genes that display unique expression patterns in embryonic bat wings and feet, compared with mouse fore- and hindlimbs. The expression of all 5'HoxD genes (Hoxd9-13) and Tbx3, six known crucial transcription factors for limb and digit development, is extremely high and prolonged in the elongating wing area. The expression of Fam5c, a tumour suppressor, in bat limbs is bat-specific and significantly high in all short digit regions (the thumb and foot digits). These results suggest multiple genetic changes occurred independently during the evolution of bat wings to elongate the hand digits, promote membrane growth and keep other digits short. Our findings also indicate that the evolution of limb morphology depends on the complex integration of multiple gene regulatory networks and biological processes that control digit formation and identity, chondrogenesis, and interdigital regression or retention.

Jointed tails enhance control of three-dimensional body rotation.

Tails used as inertial appendages induce body rotations of animals and robots-a phenomenon that is governed largely by the ratio of the body and tail moments of inertia. However, vertebrate tails have more degrees of freedom (e.g., number of joints, rotational axes) than most current theoretical models and robotic tails. To understand how morphology affects inertial appendage function, we developed an optimization-based approach that finds the maximally effective tail trajectory and measures error from a target trajectory. For tails of equal total length and mass, increasing the number of equal-length joints increased the complexity of maximally effective tail motions. When we optimized the relative lengths of tail bones while keeping the total tail length, mass, and number of joints the same, this optimization-based approach found that the lengths match the pattern found in the tail bones of mammals specialized for inertial maneuvering. In both experiments, adding joints enhanced the performance of the inertial appendage, but with diminishing returns, largely due to the total control effort constraint. This optimization-based simulation can compare the maximum performance of diverse inertial appendages that dynamically vary in moment of inertia in 3D space, predict inertial capabilities from skeletal data, and inform the design of robotic inertial appendages.