Chemically stable fluorescent proteins for advanced microscopy.

We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.

Preseptal transconjunctival approach in orbital floor fracture repairs to prevent postoperative lower eyelid malposition.

PURPOSE: Lower eyelid malposition can be a complication following orbital floor fracture surgeries. We present our incidence of lower eyelid malposition from a large case series of orbital floor fracture repairs using the 'swinging eyelid' approach and 'hang back' technique. METHODS: A retrospective review of all orbital fracture surgeries at our institution from November 2011 to March 2021 was performed. Primary outcomes included the incidence of lower eyelid malposition by category, the average time to presentation after primary surgery, and reoperation rates among cases with lower eyelid complications. RESULTS: A total of 438 cases that involved repair of the fractured orbital floor were identified. Six patients (1.37 %) developed lower eyelid malposition following primary orbital floor repair. Two patients (0.46 %) developed reverse ptosis of the lower eyelid. Two patients (0.46 %) returned with lower lid cicatricial ectropion. One patient (0.23 %) had postoperative lower eyelid retraction. One patient (0.23 %) had postoperative lower eyelid cicatricial entropion. No cases of lower lid flattening, lower eyelid fat flattening, or eyelid notch was noted. All patients with lower eyelid malposition underwent additional surgeries except one patient with reverse ptosis (83.3 %). The average time to the presentation of postoperative complications from the surgery date was 292.8 days (range = 49 days to 3.5 years). CONCLUSION: Lower eyelid malposition after orbital floor repair is a known complication that can be decreased by employing the 'swinging eyelid' with a preseptal approach and closure by the 'hang back' technique.

mGreenLantern: a bright monomeric fluorescent protein with rapid expression and cell filling properties for neuronal imaging.

Although ubiquitous in biological studies, the enhanced green and yellow fluorescent proteins (EGFP and EYFP) were not specifically optimized for neuroscience, and their underwhelming brightness and slow expression in brain tissue limits the fidelity of dendritic spine analysis and other indispensable techniques for studying neurodevelopment and plasticity. We hypothesized that EGFP's low solubility in mammalian systems must limit the total fluorescence output of whole cells, and that improving folding efficiency could therefore translate into greater brightness of expressing neurons. By introducing rationally selected combinations of folding-enhancing mutations into GFP templates and screening for brightness and expression rate in human cells, we developed mGreenLantern, a fluorescent protein having up to sixfold greater brightness in cells than EGFP. mGreenLantern illuminates neurons in the mouse brain within 72 h, dramatically reducing lag time between viral transduction and imaging, while its high brightness improves detection of neuronal morphology using widefield, confocal, and two-photon microscopy. When virally expressed to projection neurons in vivo, mGreenLantern fluorescence developed four times faster than EYFP and highlighted long-range processes that were poorly detectable in EYFP-labeled cells. Additionally, mGreenLantern retains strong fluorescence after tissue clearing and expansion microscopy, thereby facilitating superresolution and whole-brain imaging without immunohistochemistry. mGreenLantern can directly replace EGFP/EYFP in diverse systems due to its compatibility with GFP filter sets, recognition by EGFP antibodies, and excellent performance in mouse, human, and bacterial cells. Our screening and rational engineering approach is broadly applicable and suggests that greater potential of fluorescent proteins, including biosensors, could be unlocked using a similar strategy.

Sex differences in adipose development in a hunter-gatherer population.

OBJECTIVE: Humans are unusually sexually dimorphic in body composition, with adult females having on average nearly twice the fat mass as males. The development of adipose sex differences has been well characterized for children growing up in food-abundant environments, with less known about cross-cultural variation, particularly in populations without exposure to market foods, mechanized technologies, schooling, vaccination, or other medical interventions. METHODS: To add to the existing cross-cultural data, we fit multiple growth curves to body composition and anthropometric data to describe adipose development for the Savanna Pumé, South American hunter-gatherers. RESULTS: (1) Little evidence is found for an adiposity 'rebound' at the end of early childhood among either Savanna Pumé girls or boys. (2) Rather, fat deposition fluctuates during childhood, from age ~4 to ~9 years, with no appreciable accumulation until the onset of puberty, a pattern also observed among Congo Baka hunter-gatherers. (3) Body fat fluctuations are more pronounced for girls than boys. (4) The age of peak skeletal, weight, and adipose gains are staggered to a much greater extent among the Savanna Pumé compared to the National Health and Nutrition Examination Survey (NHANES III) reference, suggesting this is an important developmental strategy in lean populations. CONCLUSION: Documenting growth patterns under diverse preindustrial energetic conditions provides an important baseline for understanding sex differences in body fat emerging today under food abundance.

Wraparound nylon foil implant for isolated orbital floor fractures.

PURPOSE: There are a variety of implant materials available for orbital floor fracture repair. Implant selection is guided by surgeon experience, availability, and patient specific needs. The purpose of this study is to describe a "wraparound" technique for nylon foil implant placement for large, isolated floor fractures that provides excellent results with low incidence of enophthalmos or other complications. MATERIALS AND METHODS: A retrospective chart review from 2012 to 2020 was conducted in patients who underwent isolated orbital floor fracture repair with the use of the "wraparound" nylon foil implant. The surgical technique is described. Preoperative CT scans were assessed, and the patients were divided into groups based on the size of the floor fracture. Postoperative data was collected including Hertel measurements and complications related to the implant. RESULTS: There were eighty patients who underwent orbital floor fracture repair with the described technique and had adequate follow-up. There were 18 (22.5%) small-sized fractures, 32 (40%) medium-sized fractures, and 30 (37.5%) large-sized fractures in the study group. One patient (3.33%) in the large fracture group had clinically significant enophthalmos of 2 mm postoperatively. There were no other patients with clinically significant enophthalmos. There were no instances of any complications related to the implant, and no patients required implant removal. CONCLUSIONS: The "wraparound" technique for a nylon foil implant provides excellent results for isolated orbital floor fractures. It provides more support and stability than traditional nylon implants for larger fractures and has minimal complication rates.

Unique orbital biphenotypic tumour with neuroendocrine and sebaceous carcinoma features.

A 60-year-old female presented with a large, left upper eyelid mass that had rapidly expanded in the 3 months prior to presentation. She had a presumed chalazion excised from the same area 1.5 years ago, but no pathology was investigated. On examination, she had a palpebral mass measuring 4.5 cm x 3.5 cm that abutted the globe with extensive conjunctival involvement. Neuroimaging demonstrated lesions concerning for parotid gland metastases. An incisional biopsy demonstrated synaptophysin-positive small blue cells concerning for neuroendocrine carcinoma. The patient underwent orbital exenteration with parotidectomy and radical neck dissection. The excised mass was found to have distinct neuroendocrine carcinoma cells intermingled with sebaceous carcinoma cells, a combination not previously reported.

Ocular motility and diplopia measurements following orbital floor fracture repair.

PURPOSE: Diplopia and ocular motility restriction following orbital fracture repair are common complications. The reported rates in the literature differ greatly, in part due to varying definitions of diplopia and methods of measurement. The purpose of this study is to describe a practical and efficient in-office method for examining ocular motility and diplopia in orbital trauma patients and to report the outcomes in a series of patients who underwent orbital floor fracture repair. MATERIALS AND METHODS: A retrospective chart review from 2012 to 2019 was conducted in patients who underwent isolated orbital floor fracture repair within 3 weeks of trauma. All patients had examinations to assess extraocular motility and subjective diplopia using the described techniques. RESULTS: Ninety-three patients underwent orbital floor fracture repair and had adequate follow-up. Preoperatively, 71 (76%) patients had some restriction in motility and 59 (63%) patients complained of diplopia. Postoperatively, only 1 patient (1.09%) had clinically significant diplopia. Five (5.4%) additional patients demonstrated mild restriction in supraduction upon detailed ophthalmic examination that was not discovered upon subjective history. No patients had worsening of diplopia or motility after surgery. CONCLUSIONS: Diplopia and motility restriction following orbital fracture repair can be a persistent problem for some patients. It is important to perform a careful ophthalmic examination to detect motility deficits and diplopia that can be significant to the patient. The true rate of restriction and diplopia may be higher using detailed ophthalmic diagnostic techniques compared to subjective patient history.

Ophthalmic Surgeries and Systemic Medical Conditions Associated With the Development of Involutional Ptosis.

PURPOSE: To investigate the associations between systemic medical conditions and types of ocular surgery and the development of aponeurotic ptosis. METHODS: In this case-control observational cohort study, the relative prevalence of involutional ptosis in a large population of adult patients was assessed. Deidentified patient data from a 10-year time period was obtained from the electronic medical records of 5 large academic medical centers. Patients were selected based on ICD9 and ICD10 codes for involutional ptosis. Control patients were age and gender matched and randomly selected from a general adult population. Systemic comorbidities were determined based on ICD9 and ICD10 codes, and prior ocular surgeries based on CPT codes. The influence of systemic comorbidities and ocular surgery was examined utilizing logistic regression analysis. RESULTS: The study cohort consisted of 8297 adult patients with involutional blepharoptosis and 13,128 matched controls. The average age was 65 years. The 3 significant risk factors for developing ptosis were ocular surgery, hyperthyroidism, and type II diabetes with odds ratios of 4.2, 2.5, and 1.45, respectively (p < 0.05). Strabismus, cornea, and glaucoma surgeries were more highly associated with developing ptosis (p < 0.05). Strabismus surgery had the greatest odds ratio of 3.37, followed by cornea surgery at 2.31, and glaucoma surgery at 1.56. CONCLUSIONS: Involutional ptosis is likely a multifactorial process. This study demonstrates that risk factors for the development of ptosis include ocular surgery, specifically strabismus, cornea, and glaucoma surgery, as well as hyperthyroidism and type II diabetes.

Changes in Intraocular Pressure During Orbital Floor Fracture Repair.

PURPOSE: To evaluate changes in intraocular pressure (IOP) during orbital floor fracture repair. METHODS: Retrospective chart review of patients undergoing isolated orbital floor fracture repair with implant placement at a level 1 trauma center and tertiary care center. Patients with combined zygomaticomaxillary or medial wall fracture repair, or other significant ocular injuries were excluded. IOP measurements using a handheld applanation tonometer were recorded immediately after induction of anesthesia, immediately after orbital floor implant placement, and on postoperative day 1. RESULTS: Nine patients who underwent isolated orbital floor fracture repair were examined. There was a statistically significant mean decrease of 4.66 mmHg (p < 0.05) in IOP from prior to surgical to immediately after implant placement. There was also a significant mean increase in IOP of 7.44 mmHg (p < 0.05) at postoperative day 1 compared with immediately after implant placement. There was no statistically significant difference in IOP between prior to surgical incision and postoperative day 1. CONCLUSIONS: All patients who underwent orbital floor fracture repair in this study had a significant decrease in IOP during orbital floor fracture repair that returned to immediately preoperative levels by postoperative day 1. This initial decrease in IOP may be because of intraoperative manipulation of the globe.

Development of adiposity among Ju/'Hoansi Hunter-Gatherers.

OBJECTIVES: To characterize the development of childhood adiposity among the Ju'/Hoansi, a well-known hunter-gather group, to compare our results to U.S. references and to recently published results from Savanna Pume' foragers of Venezuela, with the goal of expanding our understanding of adipose development among human hunter-gatherers. METHODS: Triceps, subscapular, and abdominal skinfolds, along with height and weight from ~120 Ju'/Hoansi girls and ~103 boys, ages 0 to 24 years, collected in 1967-1969 were analyzed using best-fit polynomial models and penalized spines to characterize age-specific patterns of adiposity and their relationship to changes in height and weight. RESULTS: Overall, Ju/'Hoansi boys and girls exhibit small skinfolds with a decline in adiposity from 3 to 10 years, with no consistent differences among the three skinfolds. Increases in adiposity during adolescence precede peak height and weight velocities. Adiposity declines during young adulthood for girls and remains largely constant for boys. DISCUSSION: Compared to U.S. standards, the Ju/'Hoansi show a strikingly different pattern of adipose development, including the lack of an adiposity rebound at the onset of middle childhood, and clear increases in adiposity only at adolescence. These findings are consistent with published results from the Savanna Pumé hunter-gatherers of Venezuela, a group with a very different selective history, suggesting that the adiposity rebound does not characterize hunter-gathering populations more generally. Similar analyses in other subsistence populations are called for to confirm our results, and help distinguish the impact of specific environmental and dietary factors on adipose development.

Brain-wide analysis of the supraspinal connectome reveals anatomical correlates to functional recovery after spinal injury.

The supraspinal connectome is essential for normal behavior and homeostasis and consists of numerous sensory, motor, and autonomic projections from brain to spinal cord. Study of supraspinal control and its restoration after damage has focused mostly on a handful of major populations that carry motor commands, with only limited consideration of dozens more that provide autonomic or crucial motor modulation. Here, we assemble an experimental workflow to rapidly profile the entire supraspinal mesoconnectome in adult mice and disseminate the output in a web-based resource. Optimized viral labeling, 3D imaging, and registration to a mouse digital neuroanatomical atlas assigned tens of thousands of supraspinal neurons to 69 identified regions. We demonstrate the ability of this approach to clarify essential points of topographic mapping between spinal levels, measure population-specific sensitivity to spinal injury, and test the relationships between region-specific neuronal sparing and variability in functional recovery. This work will spur progress by broadening understanding of essential but understudied supraspinal populations.

In the company of men: quality of life and social support among the Ariaal of northern Kenya.

To determine the age-related pattern of well-being among men and its predictors in a subsistence society, we collected anthropometric and questionnaire data among Ariaal pastoral nomads of northern Kenya. The sample consisted of 102 settled and 103 nomadic men ages 20 to 60+ stratified by 10 year age groups. Measures included questions from the WHO quality of life questionnaire (WHOQOL), anthropometrics, and hand grip strength, as well as questions about the number of friends and kin who gave material and emotional support. Results show that while reported quality of life declined significantly across age groups in both sub-populations, nomadic males reported significantly higher overall quality of life than did settled males. Support from other males, but not marital status, was a significant predictor of quality of life, controlled for age group and residence. Among the physical measures, % body fat was positively related to quality of life among the nomads, while grip strength was not related to quality of life. In a multivariate model, male support was the strongest predictor of quality of life. These results provide the first clear demonstration of age-related declines in male quality of life and the importance of social support to men's quality of life in a subsistence population.

Androgen receptor CAG repeats and body composition among Ariaal men.

To determine the population variation in the androgen receptor (AR) and its association with body composition in a subsistence population, we sampled 87 settled and 65 nomadic males ages 20+ among the Ariaal of northern Kenya. Anthropometric measures included height, body mass index, fat-free mass (FFM), upper arm muscle plus bone area (AMPBA), % body fat (%BF), suprailliac skinfold (SISF), and waist-to-hip ratio. Salivary testosterone (T) was determined from both morning (Am T) and afternoon (Pm T) samples. Hair roots were obtained for genotyping AR CAG repeat length. AR CAG repeat length did not vary between the two sub-groups (overall value = 22.6 +/- 3.1). Multiple regression models, controlling for age and residence, indicate that Pm T was positively associated with all measures of body composition. AR CAG repeat length was a significant positive predictor of height, FFM, %BF, SISF and waist circumference. There was a significant negative Pm T by AR CAG repeat length interaction in predicting all anthropometric measures but AMPBA. These findings provide evidence for population variation in AR CAG repeat length and suggest that both T and AR CAG length play a role in body composition in this extremely lean population.

Sex-specific contribution of DHEA-cortisol ratio to prefrontal-hippocampal structural development, cognitive abilities and personality traits.

Although dehydroepiandrosterone (DHEA) may exert neuroprotective effects in the developing brain, prolonged or excessive elevations in cortisol may exert neurotoxic effects. The ratio between DHEA and cortisol (DC ratio) has been linked to internalising and externalising disorders, as well as cognitive performance, supporting the clinical relevance of this hormonal ratio during development. However, the brain mechanisms by which these effects may be mediated have not yet been identified. Furthermore, although there is evidence that the effects of cortisol in the central nervous system may be sexually dimorphic in humans, the opposite is true for DHEA, with human studies showing no sex-specific associations in cortical thickness, cortico-amygdalar or cortico-hippocampal structural covariance. Therefore, it remains unclear whether sex moderates the developmental associations between DC ratio, brain structure, cognition and behaviour. In the present study, we examined the associations between DC ratio, structural covariance of the hippocampus with whole-brain cortical thickness, and measures of personality, behaviour and cognition in a longitudinal sample of typically developing children, adolescents and young adults aged 6-22 years (N = 225 participants [F = 128]; 355 scans [F = 208]), using mixed effects models that accounted for both within- and between-subject variances. We found sex-specific interactions between DC ratio and anterior cingulate cortex-hippocampal structural covariance, with higher DC ratios being associated with a more negative covariance between these structures in girls, and a more positive covariance in boys. Furthermore, the negative prefrontal-hippocampal structural covariance found in girls was associated with higher verbal memory and mathematical ability, whereas the positive covariance found in boys was associated with lower cooperativeness and reward dependence personality traits. These findings support the notion that the ratio between DHEA and cortisol levels may contribute, at least in part, to the development of sex differences in cognitive abilities, as well as risk for internalising/externalising disorders, via an alteration in prefrontal-hippocampal structure during the transition from childhood to adulthood.

Age-specific associations between oestradiol, cortico-amygdalar structural covariance, and verbal and spatial skills.

Oestradiol is known to play an important role in the developing human brain, although little is known about the entire network of potential regions that might be affected and how these effects may vary from childhood to early adulthood, which in turn can explain sexually differentiated behaviours. In the present study, we examined the relationships between oestradiol, cortico-amygdalar structural covariance, and cognitive or behavioural measures typically showing sex differences (verbal/spatial skills, anxious-depressed symptomatology) in 152 children and adolescents (aged 6-22 years). Cortico-amygdalar structural covariance shifted from positive to negative across the age range. Oestradiol was found to diminish the impact of age on cortico-amygdalar covariance for the pre-supplementary motor area/frontal eye field and retrosplenial cortex (across the age range), as well as for the posterior cingulate cortex (in older children). Moreover, the influence of oestradiol on age-related cortico-amygdalar networks was associated with higher word identification and spatial working memory (across the age range), as well as higher reading comprehension (in older children), although it did not impact anxious-depressed symptoms. There were no significant sex effects on any of the above relationships. These findings confirm the importance of developmental timing on oestradiol-related effects and hint at the non-sexually dimorphic role of oestradiol-related cortico-amygdalar structural networks in aspects of cognition distinct from emotional processes.

Crystal Structure of Green Fluorescent Protein Clover and Design of Clover-Based Redox Sensors.

We have determined the crystal structure of Clover, one of the brightest fluorescent proteins, and found that its T203H/S65G mutations relative to wild-type GFP lock the critical E222 side chain in a fixed configuration that mimics the major conformer of that in EGFP. The resulting equilibrium shift to the predominantly deprotonated chromophore increases the extinction coefficient (EC), opposes photoactivation, and is responsible for the bathochromic shift. Clover's brightness can further be attributed to a π-π stacking interaction between H203 and the chromophore. Consistent with these observations, the Clover G65S mutant reversed the equilibrium shift, dramatically decreased the EC, and made Clover photoactivatable under conditions that activated photoactivatable GFP. Using the Clover structure, we rationally engineered a non-photoactivatable redox sensor, roClover1, and determined its structure as well as that of its parental template, roClover0.1. These high-resolution structures provide deeper insights into structure-function relationships in GFPs and may aid the development of excitation-improved ratiometric biosensors.

Mitochondrial Aging Defects Emerge in Directly Reprogrammed Human Neurons due to Their Metabolic Profile.

Mitochondria are a major target for aging and are instrumental in the age-dependent deterioration of the human brain, but studying mitochondria in aging human neurons has been challenging. Direct fibroblast-to-induced neuron (iN) conversion yields functional neurons that retain important signs of aging, in contrast to iPSC differentiation. Here, we analyzed mitochondrial features in iNs from individuals of different ages. iNs from old donors display decreased oxidative phosphorylation (OXPHOS)-related gene expression, impaired axonal mitochondrial morphologies, lower mitochondrial membrane potentials, reduced energy production, and increased oxidized proteins levels. In contrast, the fibroblasts from which iNs were generated show only mild age-dependent changes, consistent with a metabolic shift from glycolysis-dependent fibroblasts to OXPHOS-dependent iNs. Indeed, OXPHOS-induced old fibroblasts show increased mitochondrial aging features similar to iNs. Our data indicate that iNs are a valuable tool for studying mitochondrial aging and support a bioenergetic explanation for the high susceptibility of the brain to aging.

Dehydroepiandrosterone impacts working memory by shaping cortico-hippocampal structural covariance during development.

Existing studies suggest that dehydroepiandrosterone (DHEA) may be important for human brain development and cognition. For example, molecular studies have hinted at the critical role of DHEA in enhancing brain plasticity. Studies of human brain development also support the notion that DHEA is involved in preserving cortical plasticity. Further, some, though not all, studies show that DHEA administration may lead to improvements in working memory in adults. Yet these findings remain limited by an incomplete understanding of the specific neuroanatomical mechanisms through which DHEA may impact the CNS during development. Here we examined associations between DHEA, cortico-hippocampal structural covariance, and working memory (216 participants [female=123], age range 6-22 years old, mean age: 13.6 +/-3.6 years, each followed for a maximum of 3 visits over the course of 4 years). In addition to administering performance-based, spatial working memory tests to these children, we also collected ecological, parent ratings of working memory in everyday situations. We found that increasingly higher DHEA levels were associated with a shift toward positive insular-hippocampal and occipito-hippocampal structural covariance. In turn, DHEA-related insular-hippocampal covariance was associated with lower spatial working memory but higher overall working memory as measured by the ecological parent ratings. Taken together with previous research, these results support the hypothesis that DHEA may optimize cortical functions related to general attentional and working memory processes, but impair the development of bottom-up, hippocampal-to-cortical connections, resulting in impaired encoding of spatial cues.