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Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale 1 . However, the energetics driving folding are invisible in these structures and remain largely unknown 2 . The hidden thermodynamics of folding can drive disease 3,4 , shape protein evolution 5-7 and guide protein engineering 8-10 , and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability., (© 2023. The Author(s).)

Until now, membrane-protein stabilization has relied on iterations of mutations and screening. We now validate a one-step algorithm, mPROSS, for stabilizing membrane proteins directly from an AlphaFold2 model structure. Applied to the lipid-generating enzyme, ceramide synthase, 37 designed mutations lead to a more stable form of human CerS2. Together with molecular dynamics simulations, we propose a pathway by which substrates might be delivered to the ceramide synthases., (© 2023. The Author(s).)

Patients with schizophrenia have a high prevalence of cigarette smoking and respond poorly to conventional treatments, highlighting the need for new therapies. We conducted a mechanistic, proof-of-concept study using bilateral deep repetitive transcranial magnetic stimulation (dTMS) of insular and prefrontal cortices at high frequency, using the specialized H4 coil. Feasibility of dTMS was tested for disruption of tobacco self-administration, insula target engagement, and insula circuit modulation, all of which were a priori outcomes of interest. Twenty patients completed the study, consisting of weekday dTMS sessions (randomization to active dTMS or sham; double-blind; 10 patients per group), a laboratory tobacco self-administration paradigm (pre/post assessments), and multimodal imaging (three MRI total sessions). Results showed that participants assigned to active dTMS were slower to initiate smoking their first cigarette compared with sham, consistent with smoking disruption. The imaging analyses did not reveal significant Time × Group interactions, but effects were in the anticipated directions. In arterial spin labeling analyses testing for target engagement, an overall decrease in insula blood flow, measured during a post-treatment MRI versus baseline, was numerically more pronounced in the active dTMS group than sham. In fMRI analyses, resting-state connectivity between the insula and default mode network showed a numerically greater change from baseline in the active dTMS group than sham, consistent with a functional change to insula circuits. Exploratory analyses further suggested a therapeutic effect of dTMS on symptoms of psychosis. These initial observations pave the way for future confirmatory studies of dTMS in smoking patients with schizophrenia., (© 2022. The Author(s).)

Stabilizing antigenic proteins as vaccine immunogens or diagnostic reagents is a stringent case of protein engineering and design as the exterior surface must maintain recognition by receptor(s) and antigen-specific antibodies at multiple distinct epitopes. This is a challenge, as stability enhancing mutations must be focused on the protein core, whereas successful computational stabilization algorithms typically select mutations at solvent-facing positions. In this study, we report the stabilization of SARS-CoV-2 Wuhan Hu-1 Spike receptor binding domain using a combination of deep mutational scanning and computational design, including the FuncLib algorithm. Our most successful design encodes I358F, Y365W, T430I, and I513L receptor binding domain mutations, maintains recognition by the receptor ACE2 and a panel of different anti-receptor binding domain monoclonal antibodies, is between 1 and 2°C more thermally stable than the original receptor binding domain using a thermal shift assay, and is less proteolytically sensitive to chymotrypsin and thermolysin than the original receptor binding domain. Our approach could be applied to the computational stabilization of a wide range of proteins without requiring detailed knowledge of active sites or binding epitopes. We envision that this strategy may be particularly powerful for cases when there are multiple or unknown binding sites., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Summary: Many natural and designed proteins are only marginally stable limiting their usefulness in research and applications. Recently, we described an automated structure and sequence-based design method, called PROSS, for optimizing protein stability and heterologous expression levels that has since been validated on dozens of proteins. Here, we introduce improvements to the method, workflow and presentation, including more accurate sequence analysis, error handling and automated analysis of the quality of the sequence alignment that is used in design calculations., Availability and Implementation: PROSS2 is freely available for academic use at https://pross.weizmann.ac.il., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Neuromelanin-sensitive MRI (NM-MRI) purports to detect the content of neuromelanin (NM), a product of dopamine metabolism that accumulates with age in dopamine neurons of the substantia nigra (SN). Interindividual variability in dopamine function may result in varying levels of NM accumulation in the SN; however, the ability of NM-MRI to measure dopamine function in nonneurodegenerative conditions has not been established. Here, we validated that NM-MRI signal intensity in postmortem midbrain specimens correlated with regional NM concentration even in the absence of neurodegeneration, a prerequisite for its use as a proxy for dopamine function. We then validated a voxelwise NM-MRI approach with sufficient anatomical sensitivity to resolve SN subregions. Using this approach and a multimodal dataset of molecular PET and fMRI data, we further showed the NM-MRI signal was related to both dopamine release in the dorsal striatum and resting blood flow within the SN. These results suggest that NM-MRI signal in the SN is a proxy for function of dopamine neurons in the nigrostriatal pathway. As a proof of concept for its clinical utility, we show that the NM-MRI signal correlated to severity of psychosis in schizophrenia and individuals at risk for schizophrenia, consistent with the well-established dysfunction of the nigrostriatal pathway in psychosis. Our results indicate that noninvasive NM-MRI is a promising tool that could have diverse research and clinical applications to investigate in vivo the role of dopamine in neuropsychiatric illness., Competing Interests: The authors declare no conflict of interest.

Recent genetic, molecular and post-mortem studies suggest impaired dopamine (DA)-D2 receptor (D2R) trafficking in patients with schizophrenia (SZ). Imaging and preclinical studies have shown agonist-induced D2R internalization can be imaged with positron emission tomography (PET) using D2R radiotracers combined with psychostimulant challenge. This is feasible if radiotracer binding is measured when postchallenge DA levels have returned to baseline, following the initial competition phase between DA and radiotracer for binding to D2R. Here we used 'late-phase' imaging after challenge to test the hypothesis that impaired D2R internalization in SZ leads to blunted late-phase displacement, or a faster return to baseline, in patients compared with healthy controls (HCs). We imaged 10 patients with SZ and 9 HCs with PET and [ 11 C]raclopride at baseline and two times (3-5 and 6-10 h) following 0.5 mg kg -1 dextroamphetamine. We measured binding potential relative to non-displaceable compartment (BP ND ) and derived percent reduction from baseline (ΔBP ND ) for each postamphetamine scan. To test the hypothesis that time course of return of striatal BP ND to baseline differed between SZ and HCs, we implemented a linear model with ΔBP ND as dependent variable, time after amphetamine as repeated measure and time after amphetamine and diagnostic group as fixed effects. Neither diagnostic group nor interaction of diagnostic group-by-time after amphetamine significantly affected striatal ΔBP ND (F=1.38, P=0.26; F=0.51, P=0.61). These results show similar pattern of return of BP ND to baseline as a function of time in patients with SZ and HC, suggesting that striatal D2R internalization as measured by our imaging paradigm is normal in patients with SZ.

Hallucinations, a cardinal feature of psychotic disorders such as schizophrenia, are known to depend on excessive striatal dopamine. However, an underlying cognitive mechanism linking dopamine dysregulation and the experience of hallucinatory percepts remains elusive. Bayesian models explain perception as an optimal combination of prior expectations and new sensory evidence, where perceptual distortions such as illusions and hallucinations may occur if prior expectations are afforded excessive weight. Such excessive weight of prior expectations, in turn, could stem from a gain-control process controlled by neuromodulators such as dopamine. To test for such a dopamine-dependent gain-control mechanism of hallucinations, we studied unmedicated patients with schizophrenia with varying degrees of hallucination severity and healthy individuals using molecular imaging with a pharmacological manipulation of dopamine, structural imaging, and a novel task designed to measure illusory changes in the perceived duration of auditory stimuli under different levels of uncertainty. Hallucinations correlated with a perceptual bias, reflecting disproportional gain on expectations under uncertainty. This bias could be pharmacologically induced by amphetamine, strongly correlated with striatal dopamine release, and related to cortical volume of the dorsal anterior cingulate, a brain region involved in tracking environmental uncertainty. These findings outline a novel dopamine-dependent mechanism for perceptual modulation in physiological conditions and further suggest that this mechanism may confer vulnerability to hallucinations in hyper-dopaminergic states underlying psychosis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Most drugs of abuse lead to a general blunting of dopamine release in the chronic phase of dependence, which contributes to poor outcome. To test whether cannabis dependence is associated with a similar dopaminergic deficit, we examined striatal and extrastriatal dopamine release in severely cannabis-dependent participants (CD), free of any comorbid conditions, including nicotine use. Eleven CD and 12 healthy controls (HC) completed two positron emission tomography scans with [ 11 C]-(+)-PHNO, before and after oral administration of d-amphetamine. CD stayed inpatient for 5-7 days prior to the scans to standardize abstinence. Magnetic resonance spectroscopy (MRS) measures of glutamate in the striatum and hippocampus were obtained in the same subjects. Percent change in [ 11 C]-(+)-PHNO-binding potential (ΔBP ND ) was compared between groups and correlations with MRS glutamate, subclinical psychopathological and neurocognitive parameters were examined. CD had significantly lower ΔBP ND in the striatum (P=0.002, effect size (ES)=1.48), including the associative striatum (P=0.003, ES=1.39), sensorimotor striatum (P=0.003, ES=1.41) and the pallidus (P=0.012, ES=1.16). Lower dopamine release in the associative striatum correlated with inattention and negative symptoms in CD, and with poorer working memory and probabilistic category learning performance in both CD and HC. No relationships to MRS glutamate and amphetamine-induced subclinical positive symptoms were detected. In conclusion, this study provides evidence that severe cannabis dependence-without the confounds of any comorbidity-is associated with a deficit in striatal dopamine release. This deficit extends to other extrastriatal areas and predicts subclinical psychopathology., Competing Interests: Dr. Haney has received partial salary support for investigator-initiated studies from Insys Therapeutics Inc, and Lifeloc Technologies and has served as a consultant to Aelis Farma and Health Advances LLC. Dr. Kegeles has received research support from Amgen. Dr. Slifstein has received research support from Forest Laboratories, Pierre-Fabre, CHDI, and Otsuka and has provided consultation for Amgen. Dr. Abi-Dargham has received research support from Takeda and Forest Pharmaceuticals and has served on advisory boards for Roche, Forum, and Otsuka. Drs. Van de Giessen, Weinstein, Cassidy, Dong, Ghazzaoui, Ojeil, Xu, Vadhan and Volkow report no competing interests.

The energetics of membrane-protein interactions determine protein topology and structure: hydrophobicity drives the insertion of helical segments into the membrane, and positive charges orient the protein with respect to the membrane plane according to the positive-inside rule. Until recently, however, quantifying these contributions met with difficulty, precluding systematic analysis of the energetic basis for membrane-protein topology. We recently developed the dsTβL method, which uses deep sequencing and in vitro selection of segments inserted into the bacterial plasma membrane to infer insertion-energy profiles for each amino acid residue across the membrane, and quantified the insertion contribution from hydrophobicity and the positive-inside rule. Here, we present a topology-prediction algorithm called TopGraph, which is based on a sequence search for minimum dsTβL insertion energy. Whereas the average insertion energy assigned by previous experimental scales was positive (unfavorable), the average assigned by TopGraph in a nonredundant set is -6.9 kcal/mol. By quantifying contributions from both hydrophobicity and the positive-inside rule we further find that in about half of large membrane proteins polar segments are inserted into the membrane to position more positive charges in the cytoplasm, suggesting an interplay between these two energy contributions. Because membrane-embedded polar residues are crucial for substrate binding and conformational change, the results implicate the positive-inside rule in determining the architectures of membrane-protein functional sites. This insight may aid structure prediction, engineering, and design of membrane proteins. TopGraph is available online (topgraph.weizmann.ac.il)., Competing Interests: The authors declare no conflict of interest.

Background: Increased left ventricular mass (LVM) is associated with adverse outcomes in patients receiving chronic hemodialysis. Among patients receiving conventional hemodialysis (CHD, 3×/week, 4 hrs/session), we evaluated whether dialysis intensification with in-centre nocturnal hemodialysis (INHD, 3×/week, 7-8 hrs/session in the dialysis unit) was associated with regression of LVM., Methods: We conducted a retrospective cohort study of CHD recipients who converted to INHD and received INHD for at least 6 months. LVM on the first echocardiogram performed at least 6 months post-conversion was compared to LVM pre-conversion. In a secondary analysis, we examined echocardiograms performed at least 12 months after starting INHD. The effect of conversion to INHD on LVM over time was also evaluated using a longitudinal analysis that incorporated all LVM data on patients with 2 or more echocardiograms., Results: Thirty-seven patients were eligible for the primary analysis. Mean age at conversion was 49 ± 12 yrs and 30% were women. Mean pre-conversion LVM was 219 ± 66 g and following conversion, LVM declined by 32 ± 58 g (p = 0.002). Among patients whose follow-up echocardiogram occurred at least 12 months following conversion, LVM declined by 40 ± 56 g (p = 0.0004). The rate of change of LVM decreased significantly from 0.4 g/yr before conversion, to -11.7 g/yr following conversion to INHD (p < 0.0001)., Conclusion: Conversion to INHD is associated with a significant regression in LVM, which may portend a more favourable cardiovascular outcome. Our preliminary findings support the need for randomized controlled trials to definitively evaluate the cardiovascular effects of INHD.