A deep-well plate enabled automated high-throughput cell line development platform.

Cell line development (CLD) plays a crucial role in the manufacturing process development of therapeutic biologics. Most biologics are produced in Chinese hamster ovary (CHO) cell. Because of the nature of random transgene integration in CHO genome and CHO's inherent plasticity, stable CHO transfectants usually have a vast diversity in productivity, growth, and product quality. Thus, we often must resort to screening a large number of cell pools and clones to increase the probability of identifying the ideal production cell line, which is a very laborious and resource-demanding process. Here we have developed a deep-well plate (DWP) enabled high throughput (DEHT) CLD platform using 24-well DWP (24DWP), liquid handler, and other automation components. This platform has capabilities covering the key steps of CLD including cell passaging, clone imaging and expansion, and fed-batch production. We are the first to demonstrate the suitability of 24DWP for CLD by confirming minimal well-to-well and plate-to-plate variability and the absence of well-to-well cross contamination. We also demonstrated that growth, production, and product quality of 24DWP cultures were comparable to those of conventional shake flask cultures. The DEHT platform enables scientists to screen five times more cultures than the conventional CLD platform, thus significantly decreases the resources needed to identify an ideal production cell line for biologics manufacturing.

Does information on age-related fertility decline and fertility policies affect university students' family and career expectations? Evidence from a randomized controlled trial.

BACKGROUND: Past research shows that young adults have poor knowledge of age-related fertility decline and that the provision of information can improve fertility knowledge. We provide university students with information on age-related fertility and fertility-related policies and investigate whether the provision of such information affects their family formation and career expectations. METHODS: A three-armed randomized controlled trial was conducted online in Singapore between September and October 2021. A total of 1000 undergraduate students were recruited through campus advertisements to complete a 30- to 45-minute online survey, which randomly exposed participants to one of three informational brochures on age-related fertility decline, fertility policies, or diabetes (control group). Participants answered questions on family formation and career expectations both before and after the information intervention. Analysis of covariance was used to assess the effects of the information intervention. RESULTS: Exposure to age-related fertility information resulted in significant reductions in the ideal age at first childbirth, significant increases in the expected probability of marriage before age 30, and (among female participants) significant increases in the expected likelihood of undergoing social egg-freezing. No difference existed in child-number ideals, educational aspirations, and income expectations between groups after exposure. No difference existed between the fertility policy information group and the control group after exposure in any of the outcomes of interest. CONCLUSIONS: Information on age-related fertility decline brought forward university students' expected timing of childbearing and marriage without reducing their educational and career expectations. The provision of fertility information at early ages, such as during university, can help correct widespread inaccurate beliefs about fertility and promote realistic family formation planning without adversely affecting educational and career goals. TRIAL REGISTRATION: ClinicalTrials.gov.

Functional and neuromuscular changes induced via a low-cost, muscle-computer interface for telerehabilitation: A feasibility study in chronic stroke.

Stroke is a leading cause of adult disability in the United States. High doses of repeated task-specific practice have shown promising results in restoring upper limb function in chronic stroke. However, it is currently challenging to provide such doses in clinical practice. At-home telerehabilitation supervised by a clinician is a potential solution to provide higher-dose interventions. However, telerehabilitation systems developed for repeated task-specific practice typically require a minimum level of active movement. Therefore, severely impaired people necessitate alternative therapeutic approaches. Measurement and feedback of electrical muscle activity via electromyography (EMG) have been previously implemented in the presence of minimal or no volitional movement to improve motor performance in people with stroke. Specifically, muscle neurofeedback training to reduce unintended co-contractions of the impaired hand may be a targeted intervention to improve motor control in severely impaired populations. Here, we present the preliminary results of a low-cost, portable EMG biofeedback system (Tele-REINVENT) for supervised and unsupervised upper limb telerehabilitation after stroke. We aimed to explore the feasibility of providing higher doses of repeated task-specific practice during at-home training. Therefore, we recruited 5 participants (age = 44-73 years) with chronic, severe impairment due to stroke (Fugl-Meyer = 19-40/66). They completed a 6-week home-based training program that reinforced activity of the wrist extensor muscles while avoiding coactivation of flexor muscles via computer games. We used EMG signals to quantify the contribution of two antagonistic muscles and provide biofeedback of individuated activity, defined as a ratio of extensor and flexor activity during movement attempt. Our data suggest that 30 1-h sessions over 6 weeks of at-home training with our Tele-REINVENT system is feasible and may improve individuated muscle activity as well as scores on standard clinical assessments (e.g., Fugl-Meyer Assessment, Action Research Arm Test, active wrist range of motion) for some individuals. Furthermore, tests of neuromuscular control suggest modest changes in the synchronization of electroencephalography (EEG) and EMG signals within the beta band (12-30 Hz). Finally, all participants showed high adherence to the training protocol and reported enjoying using the system. These preliminary results suggest that using low-cost technology for home-based telerehabilitation after severe chronic stroke is feasible and may be effective in improving motor control via feedback of individuated muscle activity.

A high-throughput screening method for evolving a demethylase enzyme with improved and new functionalities.

AlkB is a DNA/RNA repair enzyme that removes base alkylations such as N1-methyladenosine (m1A) or N3-methylcytosine (m3C) from DNA and RNA. The AlkB enzyme has been used as a critical tool to facilitate tRNA sequencing and identification of mRNA modifications. As a tool, AlkB mutants with better reactivity and new functionalities are highly desired; however, previous identification of such AlkB mutants was based on the classical approach of targeted mutagenesis. Here, we introduce a high-throughput screening method to evaluate libraries of AlkB variants for demethylation activity on RNA and DNA substrates. This method is based on a fluorogenic RNA aptamer with an internal modified RNA/DNA residue which can block reverse transcription or introduce mutations leading to loss of fluorescence inherent in the cDNA product. Demethylation by an AlkB variant eliminates the blockage or mutation thereby restores the fluorescence signals. We applied our screening method to sites D135 and R210 in the Escherichia coli AlkB protein and identified a variant with improved activity beyond a previously known hyperactive mutant toward N1-methylguanosine (m1G) in RNA. We also applied our method to O6-methylguanosine (O6mG) modified DNA substrates and identified candidate AlkB variants with demethylating activity. Our study provides a high-throughput screening method for in vitro evolution of any demethylase enzyme.

Regulation of Co-transcriptional Pre-mRNA Splicing by m6A through the Low-Complexity Protein hnRNPG.

N6-methyladenosine (m6A) modification occurs co-transcriptionally and impacts pre-mRNA processing; however, the mechanism of co-transcriptional m6A-dependent alternative splicing regulation is still poorly understood. Heterogeneous nuclear ribonucleoprotein G (hnRNPG) is an m6A reader protein that binds RNA through RRM and Arg-Gly-Gly (RGG) motifs. Here, we show that hnRNPG directly binds to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII) using RGG motifs in its low-complexity region. Through interactions with the phosphorylated CTD and nascent RNA, hnRNPG associates co-transcriptionally with RNAPII and regulates alternative splicing transcriptome-wide. m6A near splice sites in nascent pre-mRNA modulates hnRNPG binding, which influences RNAPII occupancy patterns and promotes exon inclusion. Our results reveal an integrated mechanism of co-transcriptional m6A-mediated splicing regulation, in which an m6A reader protein uses RGG motifs to co-transcriptionally interact with both RNAPII and m6A-modified nascent pre-mRNA to modulate RNAPII occupancy and alternative splicing.

Barriers, Costs, and Attitudes Toward Pediatric Cataract Surgery at Two Large Facilities in China and India.

PURPOSE: To better understand barriers of costs and attitudes toward pediatric cataract surgery in China and India. METHODS: From January 2014 to June 2015, families of children ≤ 10 years old about to undergo or having completed surgery for bilateral, non-traumatic cataract at two tertiary centers in China and India completed questionnaires regarding their demographic characteristics, financial status, living environment, health seeking behaviors, and medical burden. RESULTS: In China, 38 children (23 boys [60.5%], mean age 3.11 ± 2.88 years) were un-operated, and 44 (26 boys [59.1%], mean age 5.09 +/- 2.17 years) had undergone surgery, while in India there were 60 (44 boys [73.3%], mean age 4.61 +/- 3.32 years) and 39 (29 boys [74.4%], mean age 6.45 +/- 2.74 years) children respectively, 181 in total. Chinese children were younger at presentation (p ≤ 0.03 for both operated and un-operated) and also when cataract was detected (median [inter quartile range] 10 [3-34] versus 24 [6-60] months [p = 0.06] for un-operated, 5 [2-12] versus 36 [8-72] months [p < 0.001] for operated). Maternal education levels were lower in India (48.3% and 51.3% with elementary education only among un-operated [p = 0.11] and operated [p = 0.006] families in India versus 27.0% and 20.5% in China), as were rates of consulting medical practitioners for illness (44.7% and 36.4% for un-operated [p < 0.001] and operated [p = 0.001] in China versus 10% and 5.13% in India). CONCLUSIONS: Socioeconomic challenges to securing cataract surgery may be greater, and delays in obtaining surgery longer, in India compared to China, if these facilities are representative.

Microbiome characterization by high-throughput transfer RNA sequencing and modification analysis.

Advances in high-throughput sequencing have facilitated remarkable insights into the diversity and functioning of naturally occurring microbes; however, current sequencing strategies are insufficient to reveal physiological states of microbial communities associated with protein translation dynamics. Transfer RNAs (tRNAs) are core components of protein synthesis machinery, present in all living cells, and are phylogenetically tractable, which make them ideal targets to gain physiological insights into environmental microbes. Here we report a direct sequencing approach, tRNA-seq, and a software suite, tRNA-seq-tools, to recover sequences, abundance profiles, and post-transcriptional modifications of microbial tRNA transcripts. Our analysis of cecal samples using tRNA-seq distinguishes high-fat- and low-fat-fed mice in a comparable fashion to 16S ribosomal RNA gene amplicons, and reveals taxon- and diet-dependent variations in tRNA modifications. Our results provide taxon-specific in situ insights into the dynamics of tRNA gene expression and post-transcriptional modifications within complex environmental microbiomes.

Cognitive, Neuroanatomical, and Genetic Predictors of Executive Function in Healthy Children and Adolescents.

The Dimensional Change Card Sort (DCCS) is a measure of cognitive flexibility for children, which requires rule-use and shifting. Demographic, cognitive, regional cortical thickness, and genetic variables, including those related to language and executive function, were used to build predictive models of DCCS scores in 556 healthy pediatric participants. Gender, age, frontal, and temporal lobe regions of interest, and measures of sustained attention, inhibition, and word reading were selected as the best predictors of DCCS performance. Results indicated that DCCS performance is related to a broad range of cognitive functions and anatomic regions associated with various levels of cognitive function.

Assessing the dysexecutive syndrome in dementia.

OBJECTIVE: We compared performance on tests of dysexecutive behaviour (DB) and executive function (EF) in patients with behavioural variant frontotemporal dementia (bvFTD), primary progressive aphasia (PPA) and corticobasal syndrome (CBS). METHODS: Patients diagnosed with bvFTD (n=124), PPA (n=34) and CBS (n=85) were recruited. EF was measured with the Delis-Kaplan Executive Function System (DKEFS: performance based), and DB was measured with the Frontal Systems Behavior Scale (FrSBe: caregiver-report based). Confirmatory factor analysis characterised the relationship between EF and DB, binary logistic regression evaluated the incremental diagnostic utility of the measures and neuroimaging data from 110 patients identified neural correlates. RESULTS: EF was lowest and DB was highest in bvFTD participants. EF and DB were distinct but related (r=-0.48). Measures correctly classified 89% of bvFTD from CBS patients and 93% of bvFTD from PPA patients-30% and 13% above base rates (59%, 80%), respectively. All modalities were useful in identifying CBS and PPA, whereas DB alone was useful for identifying bvFTD. EF was uniquely associated with caudal left dorsolateral prefrontal and lateral temporo-parietal cortices. DB was uniquely associated with the cingulate (R>L), right subcallosal and right anterior frontal cortex. EF and DB were associated with the rostral dorsolateral prefrontal cortex bilaterally. CONCLUSIONS: EF and DB measures displayed criterion and construct validity, had incremental utility at low DB levels (CBS and PPA) and were associated with overlapping and distinct neural correlates. EF and DB procedures can conjointly provide useful diagnostic and descriptive information in identifying and ruling out the dysexecutive syndrome.

CSF1R mosaicism in a family with hereditary diffuse leukoencephalopathy with spheroids.

Mutations in the colony stimulating factor 1 receptor (CSF1R) have recently been discovered as causal for hereditary diffuse leukoencephalopathy with axonal spheroids. We identified a novel, heterozygous missense mutation in CSF1R [c.1990G > A p.(E664K)] by exome sequencing in five members of a family with hereditary diffuse leukoencephalopathy with axonal spheroids. Three affected siblings had characteristic white matter abnormalities and presented with progressive neurological decline. In the fourth affected sibling, early progression halted after allogeneic haematopoietic stem cell transplantation from a related donor. Blood spot DNA from this subject displayed chimerism in CSF1R acquired after haematopoietic stem cell transplantation. Interestingly, both parents were unaffected but the mother's blood and saliva were mosaic for the CSF1R mutation. Our findings suggest that expression of wild-type CSF1R in some cells, whether achieved by mosaicism or chimerism, may confer benefit in hereditary diffuse leukoencephalopathy with axonal spheroids and suggest that haematopoietic stem cell transplantation might have a therapeutic role for this disorder.

Natural history and biomarkers in hereditary sensory neuropathy type 1.

INTRODUCTION: Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is most commonly caused by missense mutations in SPTLC1. In this study we mapped symptom progression and compared the utility of outcomes. METHODS: We administered retrospective surveys of symptoms and analyzed results of nerve conduction, autonomic function testing (AFT), and PGP9.5-immunolabeled skin biopsies. RESULTS: The first symptoms were universally sensory and occurred at a median age of 20 years (range 14-54 years). The onset of weakness, ulcers, pain, and balance problems followed sequentially. Skin biopsies revealed universally absent epidermal innervation at the distal leg with relative preservation in the thigh. Neurite density was highly correlated with total Charcot-Marie-Tooth Examination Score (CMTES; r2 = -0.8) and median motor amplitude (r2 = -0.75). CONCLUSIONS: These results confirm sensory loss as the initial symptom of HSAN1 and suggest that skin biopsy may be the most promising biomarker for future clinical trials.

Altered sphingoid base profiles in type 1 compared to type 2 diabetes.

BACKGROUND: Sphingolipids are increasingly recognized to play a role in insulin resistance and diabetes. Recently we reported significant elevations of 1-deoxysphingolipids (1-deoxySL) - an atypical class of sphingolipids in patients with metabolic syndrome (MetS) and diabetes type 2 (T2DM). It is unknown whether 1-deoxySL in patients with diabetes type 1 (T1DM) are similarly elevated. FINDINGS: We analyzed the long chain base profile by LC-MS after hydrolyzing the N-acyl and O-linked headgroups in plasma from individuals with T1DM (N = 27), T2DM (N = 30) and healthy controls (N = 23). 1-deoxySLs were significantly higher in the groups with T2DM but not different between T1DM and controls. In contrast to patients with T2DM, 1-deoxSL levels are not elevated in T1DM. CONCLUSIONS: Our study indicates that the 1-deoxySL formation is not per-se caused by hyperglycemia but rather specifically associated with metabolic changes in T2DM, such as elevated triglyceride levels.

Foreign nurse importation and the supply of native nurses.

The importation of foreign registered nurses has been used as a strategy to ease nursing shortages in the United States. The effectiveness of this policy depends critically on the long-run response of native nurses. We examine the effects of immigration of foreign-born registered nurses on the long-run employment and occupational choice of native nurses. Using a variety of empirical strategies that exploit the geographical distribution of immigrant nurses across US cities, we find evidence of large displacement effects - over a ten-year period, for every foreign nurse that migrates to a city, between 1 and 2 fewer native nurses are employed in the city. We find similar results using data on nursing board exam-takers at the state level - an increase in the flow of foreign nurses significantly reduces the number of natives sitting for licensure exams in more dependent states relative to less dependent states. Using data on self-reported workplace satisfaction among a sample of California nurses, we find suggestive evidence that part of the displacement effects could be driven by a decline in the perceived quality of the workplace environment.

Hematopoietic stem cell transplantation in the leukodystrophies: a systematic review of the literature.

OBJECTIVE: The objective of this study is to systematically review the literature on worldwide numbers of leukodystrophy patients undergoing hematopoietic stem cell transplantation (HSCT) as well as the safety and efficacy of the procedure in this patient population. MATERIALS AND METHODS: A PubMed and EMBASE search up to June 2012 was conducted with a manual search of references from relevant articles. Selected studies were evaluated using internationally accepted criteria. The effect estimates of HSCT upon survival in early-stage disease versus late-stage disease were compared. RESULTS: One hundred and fifty-two studies qualified for inclusion and reported on a total of 689 patients. Study quality ranged from poor to good; no study was rated excellent. Small sample sizes limited most studies. Meta-analysis in a subset of larger studies indicates that transplantation in earlier stages of disease fairs better than in the late stages. Beyond survival, little longitudinal data on functional outcome is reported and neurological outcome is sparse. CONCLUSION: Further studies are needed to determine the neurological outcome following HSCT in the leukodystrophies. HSCT in the early stages of cerebral disease is still recommended for select leukodystrophies.

Molecular dynamics simulations of multilayer films of polyelectrolytes and nanoparticles.

We performed molecular dynamics simulations of multilayer assemblies of flexible polyelectrolytes and nanoparticles. The film was constructed by sequential adsorption of oppositely charged polymers and nanoparticles in layer-by-layer fashion from dilute solutions. We have studied multilayer films assembled from oppositely charged polyelectrolytes, oppositely charged nanoparticles, and mixed films containing both nanoparticles and polyelectrolytes. For all studied systems, the multilayer assembly proceeds through surface overcharging after completion of each deposition step. There is almost linear growth in the surface coverage and film thickness. The multilayer films assembled from nanoparticles show better layer stratification but at the same time have higher film roughness than those assembled from flexible polyelectrolytes.