Phosphomimetic substitutions in TDP-43's transiently α-helical region suppress phase separation.

Phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) is present within the aggregates of several age-related neurodegenerative disorders, such as amyotrophic lateral sclerosis, frontotemporal lobar degeneration, and Alzheimer's disease, to the point that the presence of phosphorylated TDP-43 is considered a hallmark of some of these diseases. The majority of known TDP-43 phosphorylation sites detected in amyotrophic lateral sclerosis and frontotemporal lobar degeneration patients is located in the low-complexity domain (LCD), the same domain that has been shown to be critical for TDP-43 liquid-liquid phase separation (LLPS). However, the effect of these LCD phosphorylation sites on TDP-43 LLPS has been largely unexplored, and any work that has been done has mainly focused on sites near the C-terminal end of the LCD. Here, we used a phosphomimetic approach to explore the impact of phosphorylation at residues S332 and S333, sites located within the transiently α-helical region of TDP-43 that have been observed to be phosphorylated in disease, on protein LLPS. Our turbidimetry and fluorescence microscopy data demonstrate that these phosphomimetic substitutions greatly suppress LLPS, and solution NMR data strongly suggest that this effect is at least in part due to the loss of α-helical propensity of the phosphomimetic protein variant. We also show that the S332D and S333D substitutions slow TDP-43 LCD droplet aging and fibrillation of the protein. Overall, these findings provide a biophysical basis for understanding the effect of phosphorylation within the transiently α-helical region of TDP-43 LCD on protein LLPS and fibrillation, suggesting that phosphorylation at residues 332 and 333 is not necessarily directly related to the pathogenic process.

The role of liquid-liquid phase separation in aggregation of the TDP-43 low-complexity domain.

Pathological aggregation of the transactive response DNA-binding protein of 43 kDa (TDP-43) is associated with several neurodegenerative disorders, including ALS, frontotemporal dementia, chronic traumatic encephalopathy, and Alzheimer's disease. TDP-43 aggregation appears to be largely driven by its low-complexity domain (LCD), which also has a high propensity to undergo liquid-liquid phase separation (LLPS). However, the mechanism of TDP-43 LCD pathological aggregation and, most importantly, the relationship between the aggregation process and LLPS remains largely unknown. Here, we show that amyloid formation by the LCD is controlled by electrostatic repulsion. We also demonstrate that the liquid droplet environment strongly accelerates LCD fibrillation and that its aggregation under LLPS conditions involves several distinct events, culminating in rapid assembly of fibrillar aggregates that emanate from within mature liquid droplets. These combined results strongly suggest that LLPS may play a major role in pathological TDP-43 aggregation, contributing to pathogenesis in neurodegenerative diseases.

TrkB signalling pathway mediates the protective effects of exercise in the diabetic rat retina.

Diabetic retinopathy is a leading cause of vision loss. Treatment options for early retinopathy are sparse. Exercise protects dying photoreceptors in models of retinal degeneration, thereby preserving vision. We tested the protective effects of exercise on retinal and cognitive deficits in a type 1 diabetes model and determined whether the TrkB pathway mediates this effect. Hyperglycaemia was induced in Long Evans rats via streptozotocin injection (STZ; 100 mg/kg). Following confirmed hyperglycaemia, both control and diabetic rats underwent treadmill exercise for 30 min, 5 days/week at 0 m/min (inactive groups) or 15 m/min (active groups) for 8 weeks. A TrkB receptor antagonist (ANA-12), or vehicle, was injected 2.5 h before exercise training. We measured spatial frequency and contrast sensitivity using optokinetic tracking biweekly post-STZ; retinal function using electroretinography at 4 and 8 weeks; and cognitive function and exploratory behaviour using Y-maze at 8 weeks. Retinal neurotrophin-4 was measured using ELISA. Compared with non-diabetic controls, diabetic rats showed significantly reduced spatial frequency and contrast sensitivity, delayed electroretinogram oscillatory potential and flicker implicit times and reduced cognitive function and exploratory behaviour. Exercise interventions significantly delayed the appearance of all deficits, except for exploratory behaviour. Treatment with ANA-12 significantly reduced this protection, suggesting a TrkB-mediated mechanism. Despite this, no changes in retinal neurotrohin-4 were observed with diabetes or exercise. Exercise protected against early visual and cognitive dysfunction in diabetic rats, suggesting that exercise interventions started after hyperglycaemia diagnosis may be a beneficial treatment. The translational potential is high, given that exercise treatment is non-invasive, patient controlled and inexpensive.

Enhanced recovery after surgery (ERAS) protocol in stoma reversals.

OBJECTIVE: To compare the length of hospital stay and return of bowel movement using the conventional management versus 'enhanced recovery after surgery' protocol. METHODS: This study was conducted at the Civil Hospital, Karachi, from June 2014 to May 2015, and comprised patients undergoing stoma reversal. Patients were randomly allocated in two equal groups, i.e. A (treated with conventional peri-operative management) and B (with 'enhanced recovery after surgery' protocol). Prolonged ileus, wound infection and length of hospital stay between the two groups were compared. SPSS 20 was used for statistical analysis. RESULTS: There were 60 participants who were divided into two groups of 30(50%) each. Overall, 39(65%) patients were males and 21(35%) were females. The mean age was 27.80±9.99 years in group A and 23.87±4.56 years in group B. Besides, 25(83%) patients in group A had prolonged ileus compared to 3(10.7%) in group B (p=0.00). Moreover, 14(46.7%) patients in group A and 8(26.7%) patients in group B had wound infection (p=0.10). The mean duration of hospital stay was also less in group B compared to group A (p<0.05). CONCLUSIONS: The application of 'enhanced recovery after surgery' protocol was found to be safe.

Pathological C-terminal phosphomimetic substitutions alter the mechanism of liquid-liquid phase separation of TDP-43 low complexity domain.

C-terminally phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) marks the proteinaceous inclusions that characterize a number of age-related neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration and Alzheimer's disease. TDP-43 phosphorylation at S403/S404, and especially at S409/S410, is in fact accepted as a biomarker of proteinopathy. These residues are located within the low complexity domain (LCD), which also drives the protein's liquid-liquid phase separation (LLPS). The impact of phosphorylation at these LCD sites on phase separation of the protein is a topic of great interest, as these post-translational modifications and LLPS are both implicated in proteinopathies. Here, we employed a combination of experimental and simulation-based approaches to explore this question on a phosphomimetic model of the TDP-43 LCD. Our turbidity and fluorescence microscopy data show that Ser-to-Asp substitutions at residues S403, S404, S409 and S410 alter the LLPS behavior of TDP-43 LCD. In particular, in contrast to the unmodified protein, the phosphomimetic variants display a biphasic dependence on salt concentration. Through coarse-grained modeling, we find that this biphasic salt dependence is derived from an altered mechanism of phase separation, in which LLPS-driving short-range intermolecular hydrophobic interactions are modulated by long-range attractive electrostatic interactions. Overall, this in vitro and in silico study provides a physiochemical foundation for understanding the impact of pathologically-relevant C-terminal phosphorylation on the LLPS of the TDP-43 in a more complex cellular environment.

Pathological C-terminal phosphomimetic substitutions alter the mechanism of liquid-liquid phase separation of TDP-43 low complexity domain.

C-terminally phosphorylated TAR DNA-binding protein of 43 kDa (TDP-43) marks the proteinaceous inclusions that characterize a number of age-related neurodegenerative diseases, including amyotrophic lateral sclerosis, frontotemporal lobar degeneration and Alzheimer's disease. TDP-43 phosphorylation at S403/S404 and (especially) at S409/S410 is, in fact, accepted as a biomarker of proteinopathy. These residues are located within the low complexity domain (LCD), which also drives the protein's liquid-liquid phase separation (LLPS). The impact of phosphorylation at these LCD sites on phase separation of the protein is a topic of great interest, as these post-translational modifications and LLPS are both implicated in proteinopathies. Here, we employed a combination of experimental and simulation-based approaches to explore this question on a phosphomimetic model of the TDP-43 LCD. Our turbidity and fluorescence microscopy data show that phosphomimetic Ser-to-Asp substitutions at residues S403, S404, S409 and S410 alter the LLPS behavior of TDP-43 LCD. In particular, unlike the LLPS of unmodified protein, LLPS of the phosphomimetic variants displays a biphasic dependence on salt concentration. Through coarse-grained modeling, we find that this biphasic salt dependence is derived from an altered mechanism of phase separation, in which LLPS-driving short-range intermolecular hydrophobic interactions are modulated by long-range attractive electrostatic interactions. Overall, this in vitro and in silico study provides a physiochemical foundation for understanding the impact of pathologically relevant C-terminal phosphorylation on the LLPS of TDP-43 in a more complex cellular environment.

Prevalence of Anxiety and Depression Among Women With Polycystic Ovarian Syndrome: A Cross-Sectional Study From a Tertiary Care Hospital of Islamabad, Pakistan.

Background Depression and anxiety are common psychological conditions associated with polycystic ovarian syndrome (PCOS). It is important to understand the role of various demographic and socio-economic factors that contribute to the development of these psychological conditions. Objectives The aims of this study were to determine the prevalence of anxiety and depression in women with PCOS and to find the association of various demographic and socio-economic factors with anxiety and depression. Methods This was a single-center cross-sectional study conducted at a tertiary care hospital in Islamabad, Pakistan, from May 2021 to August 2022. All female patients, aged 18 to 40 years and diagnosed with PCOS, who presented to the department of Gynecology during the study period were eligible to be enrolled in the study. The Hospital Anxiety and Depression scale (HADS) was used to determine the level of anxiety and depression in the participants. HADS comprises 14 items scored on a Likert scale ranging from 0 to 3. Seven items correspond to depression and anxiety each. The scores range from 0 to 21 for both domains. A score of 7 or less was considered normal, 8-10 as borderline, and 11 or above as abnormal for both anxiety and depression. Data were analyzed using IBM SPSS Statistics for Windows, Version 26.0 (IBM Corp., Armonk, NY, USA). Results A total of 74 patients with PCOS were included in the study. The mean age of all the participants was 26.8 ± 5.2 and the mean body mass index (BMI) was 28.7 ± 5.4. The presence of PCOS-related symptoms was observed in all 74 cases. Menstrual cycle abnormalities were the most common symptom, which was present in 57 (77.0%) cases, followed by weight gain, which was present in 50 (67.6%) cases, and hirsutism, which was present in 41 (55.4%) cases. Diabetes mellitus and hypertension were present only in three (4.1%) and two (2.7%) cases, respectively, and positive family history of depression and/or anxiety was reported by 20 (27%) cases. The mean HAD score was 7 ± 3.8 for depression and 8 ± 3.7 for anxiety. Depression was diagnosed in 13 (17.6%) cases, and anxiety was diagnosed in 15 (20.3%) cases. Depression was found to be significantly associated with BMI (p = 0.015), level of education (p = 0.033), and monthly household income (p = 0.004). Anxiety was found to be associated with employment status (p = 0.009) and current pregnancy (p = 0.007). Rest of the factors such as age, marital status, ethnicity, menstrual irregularities, comorbidities such as diabetes mellitus and hypertension, and a family history of PCOS, anxiety, or depression did not show statistically significant association with either anxiety or depression (p < 0.05). Conclusion Anxiety and depression are common in patients with PCOS. These psychological conditions are associated with various demographic and socio-economic factors such as BMI, level of education, monthly household income, employment status, and pregnancy. It is recommended to involve a multidisciplinary team while managing patients with PCOS to timely identify and treat these psychological conditions in these patients.

Sustaining Student Engagement - Successes and Challenges of a Virtual STEM Program for High School Students.

Case Western Reserve University's School of Medicine and Comprehensive Cancer Center coordinate in-depth research immersion STEM programs to engage high school students in biomedical research and encourage pursuit of careers in health-related research and clinical care. Due to COVID-19, the 2020 programs were delivered entirely virtually. Student and faculty perceptions of the virtual experience were evaluated using surveys and focus groups. Ninety percent of students felt the virtual program met expectations. Student rankings for programmatic components that could remain virtual in future years showed a preference for highly interactive activities, especially mentorship and dialogue-based activities like discussions of science in the news. Ninety-seven percent of faculty agreed students' scientific knowledge improved. Faculty commented that certain research projects (e.g., data analysis, literature reviews) were highly appropriate for a virtual program, but that the lack of hands-on laboratory activities was challenging. Increased individual attention, flexibility, and independence were hailed as strengths of the virtual program. These findings identify activities that sustain student interest in biomedical, healthcare, and cancer related research using a virtual medium and indicate mentorship and interactive discussion-based activities enhance virtual education. Moreover, the results support incorporation of interactive online pedagogical approaches to enhance student engagement virtually and in-person.

Long-Term Functional and Structural Consequences of Primary Blast Overpressure to the Eye.

Acoustic blast overpressure (ABO) injury in military personnel and civilians is often accompanied by delayed visual deficits. However, most animal model studies dealing with blast-induced visual defects have focused on short-term (≤1 month) changes. Here, we evaluated long-term (≤8 months) retinal structure and function deficits in rats with ABO injury. Adult male Long-Evans rats were subjected to ABO from a single blast (approximately 190 dB SPL, ∼63 kPa, @80 psi), generated by a shock tube device. Retinal function (electroretinography; ERG), visual function (optomotor response), retinal thickness (spectral domain-optical coherence tomography; SD-OCT), and spatial cognition/exploratory motor behavior (Y-maze) were measured at 2, 4, 6, and 8 months post-blast. Immunohistochemical analysis of glial fibrillary acidic protein (GFAP) in retinal sections was performed at 8 months post-blast. Electroretinogram a- and b-waves, oscillatory potentials, and flicker responses showed greater amplitudes with delayed implicit times in both eyes of blast-exposed animals, relative to controls. Contrast sensitivity (CS) was reduced in both eyes of blast-exposed animals, whereas spatial frequency (SF) was decreased only in ipsilateral eyes, relative to controls. Total retinal thickness was greater in both eyes of blast-exposed animals, relative to controls, due to increased thickness of several retinal layers. Age, but not blast exposure, altered Y-maze outcomes. GFAP was greatly increased in blast-exposed retinas. ABO exposure resulted in visual and retinal changes that persisted up to 8 months post-blast, mimicking some of the visual deficits observed in human blast-exposed patients, thereby making this a useful model to study mechanisms of injury and potential treatments.