An Approach to Spatiotemporally Resolve Protein Interaction Networks in Living Cells.

Cells operate through protein interaction networks organized in space and time. Here, we describe an approach to resolve both dimensions simultaneously by using proximity labeling mediated by engineered ascorbic acid peroxidase (APEX). APEX has been used to capture entire organelle proteomes with high temporal resolution, but its breadth of labeling is generally thought to preclude the higher spatial resolution necessary to interrogate specific protein networks. We provide a solution to this problem by combining quantitative proteomics with a system of spatial references. As proof of principle, we apply this approach to interrogate proteins engaged by G-protein-coupled receptors as they dynamically signal and traffic in response to ligand-induced activation. The method resolves known binding partners, as well as previously unidentified network components. Validating its utility as a discovery pipeline, we establish that two of these proteins promote ubiquitin-linked receptor downregulation after prolonged activation.

Response sub-additivity and variability quenching in visual cortex.

Sub-additivity and variability are ubiquitous response motifs in the primary visual cortex (V1). Response sub-additivity enables the construction of useful interpretations of the visual environment, whereas response variability indicates the factors that limit the precision with which the brain can do this. There is increasing evidence that experimental manipulations that elicit response sub-additivity often also quench response variability. Here, we provide an overview of these phenomena and suggest that they may have common origins. We discuss empirical findings and recent model-based insights into the functional operations, computational objectives and circuit mechanisms underlying V1 activity. These different modelling approaches all predict that response sub-additivity and variability quenching often co-occur. The phenomenology of these two response motifs, as well as many of the insights obtained about them in V1, generalize to other cortical areas. Thus, the connection between response sub-additivity and variability quenching may be a canonical motif across the cortex.

A well-oxygenated eastern tropical Pacific during the warm Miocene.

The oxygen content of the oceans is susceptible to climate change and has declined in recent decades1, with the largest effect in oxygen-deficient zones (ODZs)2, that is, mid-depth ocean regions with oxygen concentrations <5 µmol kg-1 (ref. 3). Earth-system-model simulations of climate warming predict that ODZs will expand until at least 2100. The response on timescales of hundreds to thousands of years, however, remains uncertain3-5. Here we investigate changes in the response of ocean oxygenation during the warmer-than-present Miocene Climatic Optimum (MCO; 17.0-14.8 million years ago (Ma)). Our planktic foraminifera I/Ca and δ15N data, palaeoceanographic proxies sensitive to ODZ extent and intensity, indicate that dissolved-oxygen concentrations in the eastern tropical Pacific (ETP) exceeded 100 µmol kg-1 during the MCO. Paired Mg/Ca-derived temperature data suggest that an ODZ developed in response to an increased west-to-east temperature gradient and shoaling of the ETP thermocline. Our records align with model simulations of data from recent decades to centuries6,7, suggesting that weaker equatorial Pacific trade winds during warm periods may lead to decreased upwelling in the ETP, causing equatorial productivity and subsurface oxygen demand to be less concentrated in the east. These findings shed light on how warm-climate states such as during the MCO may affect ocean oxygenation. If the MCO is considered as a possible analogue for future warming, our findings seem to support models suggesting that the recent deoxygenation trend and expansion of the ETP ODZ may eventually reverse3,4.

A human-specific modifier of cortical connectivity and circuit function.

The cognitive abilities that characterize humans are thought to emerge from unique features of the cortical circuit architecture of the human brain, which include increased cortico-cortical connectivity. However, the evolutionary origin of these changes in connectivity and how they affected cortical circuit function and behaviour are currently unknown. The human-specific gene duplication SRGAP2C emerged in the ancestral genome of the Homo lineage before the major phase of increase in brain size1,2. SRGAP2C expression in mice increases the density of excitatory and inhibitory synapses received by layer 2/3 pyramidal neurons (PNs)3-5. Here we show that the increased number of excitatory synapses received by layer 2/3 PNs induced by SRGAP2C expression originates from a specific increase in local and long-range cortico-cortical connections. Mice humanized for SRGAP2C expression in all cortical PNs displayed a shift in the fraction of layer 2/3 PNs activated by sensory stimulation and an enhanced ability to learn a cortex-dependent sensory-discrimination task. Computational modelling revealed that the increased layer 4 to layer 2/3 connectivity induced by SRGAP2C expression explains some of the key changes in sensory coding properties. These results suggest that the emergence of SRGAP2C at the birth of the Homo lineage contributed to the evolution of specific structural and functional features of cortical circuits in the human cortex.

The stabilized supralinear network accounts for the contrast dependence of visual cortical gamma oscillations.

When stimulated, neural populations in the visual cortex exhibit fast rhythmic activity with frequencies in the gamma band (30-80 Hz). The gamma rhythm manifests as a broad resonance peak in the power-spectrum of recorded local field potentials, which exhibits various stimulus dependencies. In particular, in macaque primary visual cortex (V1), the gamma peak frequency increases with increasing stimulus contrast. Moreover, this contrast dependence is local: when contrast varies smoothly over visual space, the gamma peak frequency in each cortical column is controlled by the local contrast in that column's receptive field. No parsimonious mechanistic explanation for these contrast dependencies of V1 gamma oscillations has been proposed. The stabilized supralinear network (SSN) is a mechanistic model of cortical circuits that has accounted for a range of visual cortical response nonlinearities and contextual modulations, as well as their contrast dependence. Here, we begin by showing that a reduced SSN model without retinotopy robustly captures the contrast dependence of gamma peak frequency, and provides a mechanistic explanation for this effect based on the observed non-saturating and supralinear input-output function of V1 neurons. Given this result, the local dependence on contrast can trivially be captured in a retinotopic SSN which however lacks horizontal synaptic connections between its cortical columns. However, long-range horizontal connections in V1 are in fact strong, and underlie contextual modulation effects such as surround suppression. We thus explored whether a retinotopically organized SSN model of V1 with strong excitatory horizontal connections can exhibit both surround suppression and the local contrast dependence of gamma peak frequency. We found that retinotopic SSNs can account for both effects, but only when the horizontal excitatory projections are composed of two components with different patterns of spatial fall-off with distance: a short-range component that only targets the source column, combined with a long-range component that targets columns neighboring the source column. We thus make a specific qualitative prediction for the spatial structure of horizontal connections in macaque V1, consistent with the columnar structure of cortex.

Kinetic and structural characterization of carboxyspermidine dehydrogenase of polyamine biosynthesis.

Polyamines are positively charged alkylamines ubiquitous among eukaryotes, prokaryotes, and archaea. Humans obtain polyamines through dietary intake, metabolic production, or uptake of polyamines made by gut microbes. The polyamine biosynthetic pathway used by most gut microbes differs from that used by human cells. This alternative pathway employs carboxyspermidine dehydrogenase (CASDH), an enzyme with limited characterization. Here, we solved a 1.94 Å X-ray crystal structure of Bacteroides fragilis CASDH by molecular replacement. BfCASDH is composed of three domains with a fold similar to saccharopine dehydrogenase but with a distinct active site arrangement. Using steady-state methods, we determined kcat and kcat/Km for BfCASDH and Clostridium leptum CASDH using putrescine, diaminopropane, aspartate semi-aldehyde, NADH, and NADPH as substrates. These data revealed evidence of cooperativity in BfCASDH. Putrescine is the likely polyamine substrate and NADPH is the coenzyme used to complete the reaction, forming carboxyspermidine as a product. These data provide the first kinetic characterization of CASDH-a key enzyme in the production of microbial polyamines.

Supporting parenting among Syrian refugees in Lebanon: a randomized controlled trial of the caregiver support intervention.

BACKGROUND: Parenting interventions in humanitarian settings have prioritized the acquisition of parenting knowledge and skills, while overlooking the adverse effects of stress and distress on parenting-a key mediator of refugee children's mental health. We evaluated the effectiveness of the Caregiver Support Intervention (CSI), which emphasizes caregiver wellbeing together with training in positive parenting. METHODS: We conducted a two-arm randomized controlled trial of the CSI with Syrian refugees in Lebanon, with an intent-to-treat design, from September 2019-December 2020. A total of 480 caregivers from 240 families were randomized to the CSI or a waitlist control group (1:1). Retention from baseline to endline was 93%. Data on parenting and caregiver psychological wellbeing were collected at baseline, endline, and three-month follow-up. Prospective trial registration: ISRCTN22321773. RESULTS: We did not find a significant change on overall parenting skills at endline (primary outcome endpoint) (d = .11, p = .126) or at follow-up (Cohen's d = .15, p = .054). We did find a significant effect on overall parenting skills among participants receiving the full intervention-the sub-sample not interrupted by (COVID-19) (d = 0.25, p < .05). The CSI showed beneficial effects in the full sample at endline and follow-up on harsh parenting (d = -.17, p < .05; d = .19, p < .05), parenting knowledge (d = .63, p < .001; d = .50, p < .001), and caregiver distress (d = -.33, p < .001; d = .23, p < .01). We found no effects on parental warmth and responsiveness, psychosocial wellbeing, stress, or stress management. Changes in caregiver wellbeing partially mediated the impact of the CSI on harsh parenting, accounting for 37% of the reduction in harsh parenting. CONCLUSIONS: The CSI reduced harsh parenting and caregiver distress, and demonstrated the value of addressing caregiver wellbeing as a pathway to strengthening parenting in adversity. These effects were achieved despite a pandemic-related lockdown that impacted implementation, a severe economic crisis, and widespread social unrest. Replication under less extreme conditions may more accurately demonstrate the intervention's full potential.

Improved synthesis of deoxyalpinoid B and quantification of antileishmanial activity of deoxyalpinoid B and sulforaphane.

The total synthesis and antileishmanial activity of deoxyalpinoid B is reported via a cationic gold-catalyzed Meyer-Schuster rearrangement. The activity of deoxyalpinoid B and a known inducer of oxidative stress, sulforaphane, against Leishmania donovani and Leishmania infantatum are both reported for the first time. Both compounds exhibit potent antileishmanial activity against both species. We hypothesize that the activation of intracellular oxidative stress is a key molecular response for the inhibition of Leishmania.

Comparison of gait between walking up and down an incline with a walking device in older adults.

To identify the gait differences between cane and rolling walker (RW) use on incline and how these walking devices were received and used, 26 qualified older adults walked randomly on downward and upward with a cane and a RW respectively. With the RW use, downward walking show a faster velocity, higher cadence, less stance time, bigger steps and strides than upward walking (p≤.05); but no differences were seen with cane use. When comparing walking between a RW and a cane, those using a cane had faster velocity, larger step and stride lengths (P≤.01) but only during the upward condition. Incline surface plays a critical role in gait differences when walking with walking device. Geriatric professionals need to know the changes in gait that result from the type of device being used and need to incorporate this knowledge in the education provided to older adults for proper use of a walking device.

De Novo Variants in MAPK8IP3 Cause Intellectual Disability with Variable Brain Anomalies.

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.

Efficient total synthesis of three alpinoids via the Au(I)-catalyzed Meyer-Schuster rearrangement.

The total syntheses of three structurally related natural products, deoxyalpinoid B, deoxyalpinoid A, and alpinoid F, are reported, and each features a Au(I)-catalyzed Meyer-Schuster rearrangement as the key step. The synthesis of alpinoid F is reported for the first time. The syntheses of these natural products, all of which exhibit potent anticancer activity, are readily amenable to the preparation of structural analogs.

Open-source method of image cytometry in dorsal root ganglia tissue with immunofluorescence.

Immunohistochemistry (IHC) is a valuable tool in clinical and biological research for evaluating proteins and other antigens in spatially bound tissue. In neuroinflammatory pain research, primary afferent neurons of the dorsal root ganglion (DRG) are studied to understand molecular signaling mechanisms involved in nociception (pain) and inflammation. Measuring IHC (immunofluorescence) in DRG neurons requires manual hand tracing of nuclear and somatic boundaries, which is laborious, error-prone, and may require several weeks to collect the appropriate sample size with a mouse or pen-input display monitor. To overcome these limitations and increase standardization of sampling and measurement, we employed a reliable neuronal cytoplasmic reporter, exclusive to DRG neuronal soma, in a semi-automated algorithm-based approach of Image Cytometry in rat DRG (IC-DRG). The resulting output images are binary nuclear and somatic masks of DRG neurons, defining boundaries of measurement for CellProfiler and manually scored at 94% accurate. Herein, we successfully show a novel approach of automated image analysis for DRG neurons using a robust ImageJ/FIJI script, overcoming morphological variability and imaging artifacts native to imaging frozen tissue sections processed with immunofluorescence.

Problems after flight: understanding and comparing Syrians' perspectives in the Middle East and Europe.

BACKGROUND: Syrian refugees and asylum seekers (SRAs) face multiple stressors after flight, which may vary due to different geographic, economic, cultural and socio-political contexts in the host countries. Past research has recognised the importance of participants' own perspectives. The aims of this multi-country study were to identify and compare self-reported problems of SRAs between various settings. METHODS: A semi-structured client-generated outcome measurement was used to collect data among adult SRAs in Jordan (N = 61), Turkey (N = 46) and Switzerland (N = 57) between September 2018 and November 2019. Answers were analysed following thematic analysis. RESULTS: Over half of the participants reported practical problems with an emphasis on camp-related problems (Jordan), finances (Turkey), employment (Jordan and Switzerland) and government regulations (Switzerland), followed by psychological, and social issues. CONCLUSION: This study highlights the impact of local contextual factors on wellbeing. The findings emphasise that planning preventative procedures and mental health care services for SRAs need to consider local challenges affecting the population in specific countries.

An Evaluation of Fish Tissue Monitoring Alternatives for Mercury and Selenium: Fish Muscle Biopsy Samples Versus Homogenized Whole Fillets.

Fish contaminant studies with human health protection objectives typically focus on muscle tissue, recognizing that fillets are the commonly consumed tissue fraction. Muscle biopsy punch sampling for mercury analysis has recently been used as an alternative to harvesting fish for fillets; however, there is limited information comparing fillet plug results to whole fillet results. This study was conducted to address that data gap and to test the applicability of plugs for monitoring associated with United States Environmental Protection Agency's fish tissue-based mercury and selenium water quality criteria. The mercury phase included 300 fillet homogenates and 300 field-extracted plug samples from 60 fish, and the selenium phase included 120 fillet homogenates and 120 plugs from 30 fish. Both phases showed that there were no statistically significant differences between fillet plug and homogenized fillet results at the community level; however, a selenium plug monitoring alternative must employ a sufficiently sensitive analytical method and consider total solids. Plug and fillet sampling alternatives have inherent advantages and disadvantages. Fillet sampling provides sufficient mass to consider multiple contaminants but requires fish to be harvested. Plug sampling only provides adequate mass for a single analyte but may allow fish survival, although additional research is needed on survival following plug removal.

Local microtubule organization promotes cargo transport in C. elegans dendrites.

The specific organization of the neuronal microtubule cytoskeleton in axons and dendrites is an evolutionarily conserved determinant of neuronal polarity that allows for selective cargo sorting. However, how dendritic microtubules are organized and whether local differences influence cargo transport remains largely unknown. Here, we use live-cell imaging to systematically probe the microtubule organization in Caenorhabditiselegans neurons, and demonstrate the contribution of distinct mechanisms in the organization of dendritic microtubules. We found that most non-ciliated neurons depend on unc-116 (kinesin-1), unc-33 (CRMP) and unc-44 (ankyrin) for correct microtubule organization and polarized cargo transport, as previously reported. Ciliated neurons and the URX neuron, however, use an additional pathway to nucleate microtubules at the tip of the dendrite, from the base of the cilium in ciliated neurons. Since inhibition of distal microtubule nucleation affects distal dendritic transport, we propose a model in which the presence of a microtubule-organizing center at the dendrite tip ensures correct dendritic cargo transport.

Incorporation of extracorporeal photopheresis into a reduced intensity conditioning regimen in myelodysplastic syndrome and aggressive lymphoma: results from ECOG 1402 and 1902.

BACKGROUND: Extracorporeal photopheresis (ECP) is an immunomodulatory cellular therapy which has been shown to induce a tolerogenic state in patients with acute and chronic graft-vs-host disease. ECOG-ACRIN explored the activity of ECP as a part of a reduced intensity conditioning regimen in two multicenter trials in patients with MDS (E1902) and lymphomas (E1402). While both studies closed before completing accrual, we report results in 23 patients (17 MDS and 6 lymphoma). STUDY DESIGN AND METHODS: Patients received 2 days of ECP followed by pentostatin 4 mg/m2 /day for two consecutive days, followed by 600 cGy of total body irradiation prior to stem cell infusion. Immunosuppression for aGVHD was infusional cyclosporine A or tacrolimus and methotrexate on day +1, +3, with mycophenolate mofetil starting on day 100 for chronic GVHD prophylaxis. RESULTS: All patients engrafted, with median time to neutrophil and platelet engraftment of 15-18 days and 10-18 days respectively. Grade 3 or 4 aGVHD occurred in 13% and chronic extensive GVHD in 30%. CONCLUSIONS: These studies demonstrate that ECP/pentostatin/TBI is well tolerated and associated with adequate engraftment of neutrophils and platelets in patients with lymphomas and MDS.

Experimental study of the proton-transfer reaction C + H2+ → CH+ + H and its isotopic variant (D2+).

We report absolute integral cross section (ICS) measurements using a dual-source merged-fast-beams apparatus to study the titular reactions over the relative translational energy range of Er ∼ 0.01-10 eV. We used photodetachment of C- to produce a pure beam of atomic C in the ground electronic 3P term, with statistically populated fine-structure levels. The H2+ and D2+ were formed in an electron impact ionization source, with well known vibrational and rotational distributions. The experimental work is complemented by a theoretical study of the CH2+ electronic system in the reactant and product channels, which helps to clarify the possible reaction mechanisms underlying the ICS measurements. Our measurements provide evidence that the reactions are barrierless and exoergic. They also indicate the apparent absence of an intermolecular isotope effect, to within the total experimental uncertainties. Capture models, taking into account either the charge-induced dipole interaction potential or the combined charge-quadrupole and charge-induced dipole interaction potentials, produce reaction cross sections that lie a factor of ∼4 above the experimental results. Based on our theoretical study, we hypothesize that the reaction is most likely to proceed adiabatically through the 14A' and 14A'' states of CH2+via the reaction C(3P) + H2+(2Σ+g) → CH+(3Π) + H(2S). We also hypothesize that at low collision energies only H2+(v ≤ 2) and D2+(v ≤ 3) contribute to the titular reactions, due to the onset of dissociative charge transfer for higher vibrational v levels. Incorporating these assumptions into the capture models brings them into better agreement with the experimental results. Still, for energies ⪅0.1 eV where capture models are most relevant, the modified charge-induced dipole model yields reaction cross sections with an incorrect energy dependence and lying ∼10% below the experimental results. The capture cross section obtained from the combined charge-quadrupole and charge-induced dipole model better matches the measured energy dependence but lies ∼30-50% above the experimental results. These findings provide important guidance for future quasiclassical trajectory and quantum mechanical treatments of this reaction.

Beyond the dermis-high-risk invasive squamous cell carcinoma: a retrospective review.

OBJECTIVE: Most cutaneous squamous cell carcinomas (cSCC) are low risk and can be treated with simple excision or ablation. High-risk cSCC require invasive treatment, including radical surgery. We present our experience in treating invasive cSCC of the pelvis and extremities. METHOD: A retrospective review of the data of patients with invasive cSCC, indicated for surgery between 2014 and 2018, from a single institution was carried out. RESULTS: A total of 19 patients (nine men, 10 women) were included in the study. Mean age was 62 years; mean tumour size was 8.6cm). Of the 19 patients, five patients with paraplegia with cSCC arising from hard-to-heal ulcers died of infection or bleeding after surgery or systemic therapy. Also, nine patients with localised cSCC underwent margin-negative resection with or without radiation; one patient experienced disease relapse. Of the participants, two patients with previous transplants and multifocal aggressive cSCC underwent numerous resections but succumbed to disease, and two patients who presented with locally recurrent disease after previous positive margin resection and radiation underwent re-resection but developed recurrent disease. CONCLUSIONS: Prognosis for invasive cSCC largely depends on clinical setting. Tumours arising from ulcers in patients with paraplegia have a poor prognosis regardless of treatment. Invasive cSCC in transplant patients are often multifocal and often recur. Debulking procedures are associated with local recurrence despite radiation. Patients presenting with localised disease have a favourable prognosis with wide resection, flap coverage and adjuvant therapy.

Postural effect on gait characteristics by using rolling walkers.

This study was to examine the effect of leaning-forward posture (LFP) on gait parameters while using a rolling walker (RW). A cross-sectional study was conducted in which 23 older female adults were asked to walk with a RW on the GaitRite walkway in two posture situations: upright posture, and LFP caused by pushing the RW forward and then following the RW. The temporal and spatial gait parameters were obtained for data analysis. Results showed that compared with the upright posture, participants with LFP demonstrated significantly increased cadence, decreased velocity and gait cycle time (both swing and stance time decreased). Of spatial parameters, both step and stride length significantly decreased, but the base of support increased significantly. These indicate that LFP during ambulation with a RW could lead participants to a shuffling-like (many steps on short distance) gait pattern. They may help clinicians find proper rehabilitation interventions and appropriate patient education for this specific postural presentation.