Inclusion of Sex and Gender Differences in U.S. State Action Plans for Opioid Use and Opioid Use Disorder.

Background: States have developed action plans to address the "opioid crisis" over the past several years. While sex- and gender-based differences have been identified in complications of opioid use, risks of addiction to opioids, barriers to treatment of opioid use disorder, and associated stigma, it is unknown if or to what extent opioid plans consider or account for these differences. The objectives of this study were to analyze U.S. state opioid action plans and their inclusion of sex- and gender-specific concerns. Methods: A content analysis of 49 state plans was conducted in June 2020, assessing their inclusion of 14 variables covering provider education, pregnancy-related, and sex- or gender-based differences in opioid addiction and treatment. Results: Neonatal opioid withdrawal syndrome was the most common variable, noted in 57% of plans. Only 14% included pregnancy-related stigma, and 4% identified gender-specific stigma. Contraceptives and family-planning were included in 12% and 10% of plans, respectively. Two states included more than half of the variables and five plans made no mention of sex or gender differences. Conclusions: Few state plans contained sex- or gender-specific information, and those that did focused almost exclusively on childbearing, excluding other unique considerations of opioid-using-women of all ages. The results of this study could improve the care of women using opioids by informing the strategies of state agencies and impacting legislative efforts for prevention initiatives, substance use disorder treatment, and law enforcement programs.

Viral Evolution Shaped by Host Proteostasis Networks.

Understanding the factors that shape viral evolution is critical for developing effective antiviral strategies, accurately predicting viral evolution, and preventing pandemics. One fundamental determinant of viral evolution is the interplay between viral protein biophysics and the host machineries that regulate protein folding and quality control. Most adaptive mutations in viruses are biophysically deleterious, resulting in a viral protein product with folding defects. In cells, protein folding is assisted by a dynamic system of chaperones and quality control processes known as the proteostasis network. Host proteostasis networks can determine the fates of viral proteins with biophysical defects, either by assisting with folding or by targeting them for degradation. In this review, we discuss and analyze new discoveries revealing that host proteostasis factors can profoundly shape the sequence space accessible to evolving viral proteins. We also discuss the many opportunities for research progress proffered by the proteostasis perspective on viral evolution and adaptation.

The endoplasmic reticulum proteostasis network profoundly shapes the protein sequence space accessible to HIV envelope.

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, at least in theory, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate the levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV-1) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody- and drug-escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s induction. Our data indicate that this phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly, and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is induced, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

Sarkosyl: A milder detergent than SDS for identifying proteins with moderately high hyperstability using gel electrophoresis.

Sodium dodecyl sulfate (SDS) is a detergent used as a strong denaturant of proteins in gel electrophoresis. It has previously been shown that certain hyperstable, also known as kinetically stable, proteins are resistant to SDS and thus require heating for their denaturation in the presence of SDS. Because of its high denaturing strength, relatively few proteins are resistant to SDS thereby limiting the current use of SDS-PAGE for identifying hyperstable degradation-resistant proteins. In this study, we show that sarkosyl, a milder detergent than SDS, is able to identify proteins with moderately high kinetic stability that lack SDS-resistance. Our assay involves running and subsequently comparing boiled and unheated protein samples containing sarkosyl, instead of SDS, on PAGE gels and identifying subsequent differences in protein migration. Our results also show that sarkosyl and SDS may be combined in PAGE experiments at varying relative percentages to obtain semi-quantitative information about a protein's kinetic stability in a range inaccessible by probing through native- or SDS-PAGE. Using protein extracts from various legumes as model systems, we detected proteins with a range of protein stability from nearly SDS-resistant to barely sarkosyl resistant.

Neonatal severe hyperparathyroidism caused by homozygous mutation in CASR: A rare cause of life-threatening hypercalcemia.

Neonatal severe hyperparathyroidism (NSHPT) is a rare, life-threatening condition that presents with severe hypercalcemia, hyperparathyroidism, and osteopenia in the newborn period. Treatment of NSHPT traditionally includes hydration and bisphosphonates; however newer calcimimetic agents, such as cinacalcet, are now being utilized to prevent or delay parathyroidectomy which is technically difficult in the newborn. Medical treatment success is related to calcium sensing receptor (CaSR) genotype. We report a 4-day-old infant who presented with hyperbilirubinemia, poor feeding, weight loss, severe hypotonia and was ultimately diagnosed with NSHPT. The patient's total serum calcium level of 36.8 mg/dL (reference range: 8.5-10.4 mg/dL) is, to our knowledge, the highest ever documented in this setting. Exome data previously obtained on the infant's parents was re-analyzed demonstrating bi-parental heterozygosity for a mutation of the CASR gene: c.206G > A, and Sanger sequencing data confirmed the patient was a homozygote for the same mutation. Though a patient with the same CaSR gene mutation described here has responded to cinacalcet, our patient did not respond and required parathyroidectomy. Though this case has previously been published as a surgical case report, a full report of the medical management and underlying genetic etiology is warranted; this case underscores the importance of disclosing bi-parental heterozygosity for a gene causing severe neonatal disease particularly when treatment is available and illustrates the need for further in vitro studies of this CaSR mutation.