The conceptus and its parts: o ntogenetic recapitulation in early human development.

Our understanding of the processes of human development that occur during and just after implantation is still incomplete. The anatomical studies by Erich Blechschmidt (1904-1992) at the University of Göttingen demonstrate the uniqueness and beauty of the early stages of individual human development or ontogeny. The interpretations of human embryology by Blechschmidt offer a simple unifying hypothesis: metabolic and biomechanical events are repeated, and this can be described as an ontogenetic recapitulation. This commentary provides a rationale for using some older terms and introducing new ones in the description of early human development. The product of conception is a conceptus; the outer part of the conceptus is the ectoblast and everything inside is the endoblast; the endocyst arises in the endoblast when the future amniotic fluid is forming. The human embryo arises from the innermost part of the endocyst. Terms such as morula, gastrula, and cyema, which are imported from zoology and ignore the role of the zona pellucida and constrained fluid compartments in the conceptus, can be avoided.

Establishing Order Through Disorder by the Hsp90 Molecular Chaperone.

The Heat Shock Protein 90 (Hsp90) molecular chaperone is a key driver of protein homeostasis (proteostasis) under physiologically normal and stress conditions. In eukaryotes, Hsp90 is essential and is one of the most abundant proteins in a cell where the chaperone shuttles between the cytoplasm and nucleus to fold, stabilize, and regulate client proteins and protein complexes. Numerous high-throughput screens have mapped the Hsp90 interactome, building a vast network comprising ∼25% of the proteome in budding yeast. How Hsp90 is able to associate with this diverse and large cadre of targets is critical to comprehending how the proteostatic process works. Here, we review recent progress on our understanding of the molecular underpinnings driving Hsp90-client interactions from both the perspective of the targets and Hsp90. In addition to considering the available Hsp90-client structures, we also assessed recently identified Hsp90-client peptide complexes to build a model that justifies how Hsp90 might recognize a wide spectrum of target proteins. In brief, Hsp90 either directly recognizes a site within an intrinsically disordered region (IDR) of a client protein to transiently regulate that client or it associates with an unstructured polypeptide section created by the concerted efforts of multiple chaperones and cochaperones to stably associate with a client. Overall, Hsp90 exploits a common recognition property (i.e., IDR) within diverse clients to support chaperone-actionthereby enabling its central role in proteostasis.

Use of a Candida albicans SC5314 PacBio HiFi reads dataset to close gaps in the reference genome assembly, reveal a subtelomeric gene family, and produce accurate phased allelic sequences.

Candida albicans SC5314 is the most-often used strain for molecular manipulation of the species. The SC5314 reference genome sequence is the result of considerable effort from many scientists and has advanced research into fungal biology and pathogenesis. Although the resource is highly developed and presented in a phased diploid format, the sequence includes gaps and does not extend to the telomeres on its eight chromosome pairs. Accurate SC5314 genome assembly is complicated by the presence of extensive repeated sequences and considerable allelic length variation at some loci. Advances in genome sequencing technology provide the tools to obtain highly accurate long-read data that span even the most-difficult-to-assemble genome regions. Here, we describe derivation of a PacBio HiFi data set and creation of a collapsed haploid telomere-to-telomere assembly of the SC5314 genome (ASM3268872v1) that revealed previously unknown features of the strain. ASM3268872v1 subtelomeric distances were up to 19 kb larger than in the reference genome and revealed a family of highly conserved DNA helicase-encoding genes at 10 of the 16 chromosome ends. We also describe alignments of individual HiFi reads to deduce accurate diploid sequences for the most notoriously difficult-to-assemble C. albicans genes: the agglutinin-like sequence (ALS) gene family. We provide a tutorial that demonstrates how the HiFi reads can be visualized to explore any region of interest. Availability of the HiFi reads data set and the ASM3268872v1 comparative guide assembly will streamline research efforts because accurate diploid sequences can be derived using simple in silico methods rather than time-consuming laboratory-bench approaches.

Protocol for establishing a protein interactome based on close physical proximity to a target protein within live budding yeast.

Here, we present a protocol for establishing a protein interactome based on close physical proximity to a target protein within live yeast cells. We describe steps for capturing both transient and stable binders by integrating a non-natural amino acid. We detail procedures for employing a site-directed method for labeling the surface that mediates protein associations and uncovers the binding sites on the interactors. Combined with mass spectrometry, our approach proves valuable in discovering binding partners and constructing a comprehensive protein-interaction network.

An improved Lodderomyces elongisporus NRRL YB-4239 genome assembly substantiated by its electrophoretic karyotype.

Pacific Biosciences long-read sequencing was used to improve the genome assembly for Lodderomyces elongisporus strain NRRL YB-4239 (ATCC 11503). The new assembly included eight chromosomes that were substantiated by the electrophoretic karyotype. The nuclear genome was 16.1 Mb (37.2% GC) with 5,740 genes predicted.

Safety and Efficacy of Riluzole in Acute Spinal Cord Injury Study (RISCIS): A Multi-Center, Randomized, Placebo-Controlled, Double-Blinded Trial.

Riluzole is a sodium-glutamate antagonist that attenuates neurodegeneration in amyotrophic lateral sclerosis (ALS). It has shown favorable results in promoting recovery in pre-clinical models of traumatic spinal cord injury (tSCI) and in early phase clinical trials. This study aimed to evaluate the efficacy and safety of riluzole in acute cervical tSCI. An international, multi-center, prospective, randomized, double-blinded, placebo-controlled, adaptive, Phase III trial (NCT01597518) was undertaken. Patients with American Spinal Injury Association Impairment Scale (AIS) A-C, cervical (C4-C8) tSCI, and <12 h from injury were randomized to receive either riluzole, at an oral dose of 100 mg twice per day (BID) for the first 24 h followed by 50 mg BID for the following 13 days, or placebo. The primary efficacy end-point was change in Upper Extremity Motor (UEM) scores at 180 days. The primary efficacy analyses were conducted on an intention to treat (ITT) and completed cases (CC) basis. The study was powered at a planned enrolment of 351 patients. The trial began in October 2013 and was halted by the sponsor on May 2020 (and terminated in April 2021) in the face of the global COVID-19 pandemic. One hundred ninety-three patients (54.9% of the pre-planned enrolment) were randomized with a follow-up rate of 82.7% at 180 days. At 180 days, in the CC population the riluzole-treated patients compared with placebo had a mean gain of 1.76 UEM scores (95% confidence interval: -2.54-6.06) and 2.86 total motor scores (CI: -6.79-12.52). No drug-related serious adverse events were associated with the use of riluzole. Additional pre-planned sensitivity analyses revealed that in the AIS C population, riluzole was associated with significant improvement in total motor scores (estimate: standard error [SE] 8.0; CI 1.5-14.4) and upper extremity motor scores (SE 13.8; CI 3.1-24.5) at 6 months. AIS B patients had higher reported independence, measured by the Spinal Cord Independence Measure score (45.3 vs. 27.3; d: 18.0 CI: -1.7-38.0) and change in mental health scores, measured by the Short Form 36 mental health domain (2.01 vs. -11.58; d: 13.2 CI: 1.2-24.8) at 180 days. AIS A patients who received riluzole had a higher average gain in neurological levels at 6 months compared with placebo (mean 0.50 levels gained vs. 0.12 in placebo; d: 0.38, CI: -0.2-0.9). The primary analysis did not achieve the predetermined end-point of efficacy for riluzole, likely related to insufficient power. However, on pre-planned secondary analyses, all subgroups of cervical SCI subjects (AIS grades A, B and C) treated with riluzole showed significant gains in functional recovery. The results of this trial may warrant further investigation to extend these findings. Moreover, guideline development groups may wish to assess the possible clinical relevance of the secondary outcome analyses, in light of the fact that SCI is an uncommon orphan disorder without an accepted neuroprotective treatment.

The Hsp90 molecular chaperone governs client proteins by targeting intrinsically disordered regions.

Molecular chaperones govern proteome health to support cell homeostasis. An essential eukaryotic component of the chaperone system is Hsp90. Using a chemical-biology approach, we characterized the features driving the Hsp90 physical interactome. We found that Hsp90 associated with ∼20% of the yeast proteome using its three domains to preferentially target intrinsically disordered regions (IDRs) of client proteins. Hsp90 selectively utilized an IDR to regulate client activity as well as maintained IDR-protein health by preventing the transition to stress granules or P-bodies at physiological temperatures. We also discovered that Hsp90 controls the fidelity of ribosome initiation that triggers a heat shock response when disrupted. Our study provides insights into how this abundant molecular chaperone supports a dynamic and healthy native protein landscape.

A Chromosome-Level Genome Assembly for Yamadazyma tenuis ATCC 10573.

Pacific Biosciences (PacBio) long-read sequencing was used to generate a chromosome-level genome assembly for Yamadazyma tenuis strain ATCC 10573. The assembly featured 7 chromosomes that matched the electrophoretic karyotype and a 26.5-kb circular mitochondrial genome. The nuclear genome was 10.8 Mb, with a GC content of 43%, and 5,340 predicted genes.

An Environmental Scan of Advanced Practice Radiation Therapy in the United States: A PESTEL Analysis.

PURPOSE: In 2021, the Advanced Practice Radiation Therapy Working Group (APRTWG) was established in the United States as a grassroots alliance of multidisciplinary radiation oncology professionals-radiation therapists, physicians, dosimetrists, and administrators-located across the country, interested in studying and establishing the Advanced Practice Radiation Therapist (APRT) level of practice in the United States. The APRT model has shown success in the United Kingdom, Canada, Australia, Singapore, and other countries, documenting the value of the APRT to the quality and advancement of clinical care. In the United States, the APRTWG seeks to coordinate activities, align resources, and drive the national agenda to collectively develop and define novel models of care using APRT in line with the evolving needs of patients and the radiation therapy profession. This environmental scan aims to examine the context of radiation oncology medical practice in the United States to inform pathways ahead for a proposed APRT model through a Political, Economic, Social, Technological, Environmental, and Legal (PESTEL) analysis. METHODS AND MATERIALS: A literature search was conducted to understand the chronological timeline of the development of APRT during the past 25 years. Items that included the activities, scope of practice, and implementation of APRT nationally and internationally were identified. Papers describing advanced practitioner roles that are commonly found in the multidisciplinary team in radiation oncology both in the United States and internationally, such as physician assistants and nurse practitioners, were excluded. RESULTS: Despite the environmental scan outcome, it is acknowledged that data collation and analysis was not as robust as that anticipated by undertaking a systematic review. Papers were identified by the lead author that aligned with each of the PESTEL factors. Defined broadly, a new care model can adjust how health services are delivered by incorporating best practices in patient care for a specific population, person, or patient cohort. As patients enter different stages of their disease, the purpose of a new model is to provide individuals with the right care, at the right time, by the right team, in the right place. It is clear that the opportunity for positive change and impact on the current state of practice in radiation oncology exists. CONCLUSION: The environmental scan findings demonstrate the complexities associated with implementing APRT in the United States, with multifactorial political, environmental, societal, technological, economic, and legal aspects to consider. The APRTWG will continue to lead and participate in such activities to demonstrate and identify APRT role opportunities in the United States and drive the nationwide implementation of the APRT level of practice in this country.

Second Virtual International Symposium on Cellular and Organismal Stress Responses, September 8-9, 2022.

The Second International Symposium on Cellular and Organismal Stress Responses took place virtually on September 8-9, 2022. This meeting was supported by the Cell Stress Society International (CSSI) and organized by Patricija Van Oosten-Hawle and Andrew Truman (University of North Carolina at Charlotte, USA) and Mehdi Mollapour (SUNY Upstate Medical University, USA). The goal of this symposium was to continue the theme from the initial meeting in 2020 by providing a platform for established researchers, new investigators, postdoctoral fellows, and students to present and exchange ideas on various topics on cellular stress and chaperones. We will summarize the highlights of the meeting here and recognize those that received recognition from the CSSI.

Prehospital Cardiovascular Autoregulatory Disturbances Correlate With the Functional Neuroanatomy of Acute Spinal Cord Injury.

STUDY DESIGN: Retrospective study. OBJECTIVE: The importance of attenuating the cardiovascular autoregulatory disturbances accompanying acute spinal cord injury (SCI) has long been recognized. This report assembles SCI emergency service data and correlates cardiovascular parameters to preserved functional neuroanatomy. SUMMARY OF BACKGROUND DATA: The nascent nature of evidence-based reporting of prehospital cardiovascular autoregulatory disturbances in SCI indicates the need to assemble more information. MATERIALS AND METHODS: SCI data for <24 hours were extracted from ambulance and hospital records. The mean arterial pressure (MAP) was calculated. The International Standard for Neurological Classification of SCI (ISNCSCI) evaluates the primary outcome of motor incomplete injury (grades C/D) at acute presentation. Logistic regression was adjusted for multiple confounders that were expected to influence the odds of grade C/D. RESULTS: A cohort of 99 acute SCI cases was retained; mean (SD) age 40.7±20.5 years, 88 male, 84 tetraplegic, 65 grades A/B (motor complete injury), triage time 2±1.6 hours. The lowest recorded prehospital MAP [mean (SD): 77.9±19, range: 45-145 mm Hg] approached the nadir for adequate organ perfusion. Thirty-four (52%) grade A/B and 10 (30%) C/D cases had MAP readings <85 mm Hg. In data adjusted for age, injury level, and triage time a 5 mm Hg increase in the lowest MAP value was associated with a 34% increase in the odds of having motor incomplete injury at acute presentation (adjusted odds ratio=1.34; 95% CI: 1.11-1.61; P =0.002). CONCLUSION: An important observation with implications for timely and selective cardiovascular resuscitation during SCI prehospital care involves significant negative associations between the depth of systemic hypotension and preserved functional neuroanatomy. Regardless of the mechanism, our confounder-adjusted logistic regression model extends in-hospital evidence and provides a conceptual bedside-bench framework for future investigations.

Survival Model of Thoracic Contusion Spinal Cord Injury in the Domestic Pig.

Spinal cord injury (SCI) frequently results in motor, sensory, and autonomic dysfunction for which there is currently no cure. Recent pre-clinical and clinical research has led to promising advances in treatment; however, therapeutics indicating promise in rodents have not translated successfully in human trials, likely due, in part, to gross anatomical and physiological differences between the species. Therefore, large animal models of SCI may facilitate the study of secondary injury processes that are influenced by scale, and may assist the translation of potential therapeutic interventions. The aim of this study was to characterize two severities of thoracic contusion SCI in female domestic pigs, measuring motor function and spinal cord lesion characteristics, over 2 weeks post-SCI. A custom-instrumented weight-drop injury device was used to release a 50 g impactor from 10 cm (n = 3) or 20 cm (n = 7) onto the exposed dura, to induce a contusion at the T10 thoracic spinal level. Hind limb motor function was assessed at 8 and 13 days post-SCI using a 10-point scale. Volume and extent of lesion-associated signal hyperintensity in T2-weighted magnetic resonance (MR) images were assessed at 3, 7, and 14 days post-injury. Animals were transcardially perfused at 14 days post-SCI and spinal cord tissue was harvested for histological analysis. Bowel function was retained in all animals and transient urinary retention occurred in one animal after catheter removal. All animals displayed hind limb motor deficits. Animals in the 10-cm group demonstrated some stepping and weight-bearing and scored a median 2-3 points higher on the 10-point motor function scale at 8 and 13 days post-SCI, than did the 20-cm group. Histological lesion volume was 20% greater, and 30% less white matter was spared, in the 20-cm group than in the 10-cm group. The MR signal hyperintensity in the 20-cm injury group had a median cranial-caudal extent approximately 1.5 times greater than the 10-cm injury group at all three time-points, and median volumes 1.8, 2.5, and 4.5 times greater at day 3, 7, and 14 post-injury, respectively. Regional differences in axonal injury were observed between groups, with amyloid precursor protein immunoreactivity greatest in the 20-cm group in spinal cord sections adjacent to the injury epicenter. This study demonstrated graded injuries in a domestic pig strain, with outcome measures comparable to miniature pig models of contusion SCI. The model provides a vehicle for the study of SCI and potential treatments, particularly where miniature pig strains are not available and/or where small animal models are not appropriate for the research question.

The Transcription Factor YY-1 Is an Essential Regulator of T Follicular Helper Cell Differentiation.

T follicular helper (TFH) cells are a specialized subset of CD4 T cells that deliver critical help signals to B cells for the production of high-affinity Abs. Understanding the genetic program regulating TFH differentiation is critical if one wants to manipulate TFH cells during vaccination. A large number of transcription factor (TFs) involved in the regulation of TFH differentiation have been characterized. However, there are likely additional unknown TFs required for this process. To identify new TFs, we screened a large short hairpin RNA library targeting 353 TFs in mice using an in vivo RNA interference screen. Yin Yang 1 (YY-1) was identified as a novel positive regulator of TFH differentiation. Ablation of YY-1 severely impaired TFH differentiation following acute viral infection and protein immunization. We found that the zinc fingers of YY-1 are critical to support TFH differentiation. Thus, we discovered a novel TF involved in the regulation of TFH cells.

Regulation of Protein Transport Pathways by the Cytosolic Hsp90s.

The highly conserved molecular chaperone heat shock protein 90 (Hsp90) is well-known for maintaining metastable proteins and mediating various aspects of intracellular protein dynamics. Intriguingly, high-throughput interactome studies suggest that Hsp90 is associated with a variety of other pathways. Here, we will highlight the potential impact of Hsp90 in protein transport. Currently, a limited number of studies have defined a few mechanistic contributions of Hsp90 to protein transport, yet the relevance of hundreds of additional connections between Hsp90 and factors known to aide this process remains unresolved. These interactors broadly support transport pathways including endocytic and exocytic vesicular transport, the transfer of polypeptides across membranes, or unconventional protein secretion. In resolving how Hsp90 contributes to the protein transport process, new therapeutic targets will likely be obtained for the treatment of numerous human health issues, including bacterial infection, cancer metastasis, and neurodegeneration.

Persistently Raised Serum Titanium Levels After Spinal Instrumentation in Children.

STUDY DESIGN: Prospective study of patients undergoing elective spinal deformity surgery with repeated testing for circulating metal ions including preoperative levels acting as controls. OBJECTIVE: The aim was to determine if levels of particular circulating metal ions are maintained to two years postsurgery including different implant systems and rods. SUMMARY OF BACKGROUND DATA: Adults having hip replacements, especially metal-on-metal bearings, may develop high metal ion levels. Pediatric spinal implants are known to cause circulating metal ions, notably titanium, chromium, cobalt, and nickel. MATERIALS AND METHODS: Fifty-six children having spinal deformity surgery were studied with repeated testing for circulating metal ions, using high-resolution inductively coupled plasma mass spectrometry. Linear mixed-effects models adjusting for repeated measurements over time were used to analyze levels of titanium, cobalt, chromium, and nickel. RESULTS: Titanium levels showed a rapid increase by seven days and a peak at 30 days that was essentially maintained at the two-year assay. At two years, titanium levels were 5.14 times greater compared with the presurgery control level (P<0.0001). Cobalt levels were shown to gradually rise to a peak at 30 days and then slowly decline but remained 1.74 times above mean baseline level at two years (P=0.0004), with a declining trajectory. Chromium and nickel levels rose immediately postoperatively and then steadily declined to baseline by six months and remained at baseline at two years. The five implant systems tested had generally equivalent results. CONCLUSION: The persistent and rising levels of titanium, in a predominantly female population, is concerning. Titanium is known to cross the placental barrier and enter the circulation of the fetus in rodents and humans, and to accumulate in solid organs especially the liver, spleen, heart, and lymph nodes in humans. This potentially exposes the offspring of mothers with spinal implants to titanium, with potential teratogenic effects.

Acute Cytomegalovirus Illness in an Immunocompetent Adult Causing Intravascular Hemolysis and Suspected Hemophagocytic Lymphohistiocytosis.

Background: Primary cytomegalovirus (CMV) infection of the immunocompetent host usually produces little-to-no illness. Occasionally, the infection results in mononucleosis syndrome, protracted fever, hepatitis, tissue-invasive disease, or Guillain-Barré syndrome. Hemolytic anemia and hemophagocytic lymphohistiocytosis (HLH) are rare complications that have not been reported to co-occur. Having hemolytic anemia in conjunction with more common findings of fever and hepatitis complicates the diagnosis of HLH. Case Presentation. A 34-year-old male with previously good health presented with a prolonged febrile illness, jaundice, and anemia. An extensive work-up during hospitalization revealed intravascular hemolytic anemia, leukopenia, hepatosplenomegaly, and biopsy evidence of extensive lymphohistiocytic infiltration of the liver with microgranulomata and sinusoidal hemophagocytosis. Soluble CD25 level was mildly elevated at 1200.3 pg/mL and the HScore calculation (fever, bicytopenia, hepatosplenomegaly, aspartate aminotransaminase 99 IU/L, ferritin 1570 ng/mL, fibrinogen 488 mg/dL, and triglycerides 173 mg/dL) suggested a moderate probability of reactive HLH. Primary CMV infection was diagnosed based on CMV IgM positivity, low CMV IgG avidity index, and low-grade CMV DNAemia. The CMV antigen was not detected in the liver biopsy, and the bone marrow biopsy was unremarkable. The illness began to improve before he received oral valganciclovir for 5 days, and he was in good health 10 months later. Conclusion: Acute CMV illness in an immunocompetent adult can present with hemolytic anemia and clinicopathologic abnormalities consistent with a form fruste of HLH. The illness is likely due to an excessive or unbalanced immune response that may self-correct.

Inhibiting U1 telescripting: A means to an end for transcription.

In this issue of Molecular Cell, Cugusi et al. (2022) discover that inhibition of U1 telescripting is a novel mechanism that promotes a switch between gene programs in response to heat stress.

Ethical questions arising from Otfrid Foerster's use of the Sherrington method to map human dermatomes.

Otfrid Foerster (1873-1941) is well known for his maps of human dermatomes. We have examined the history of the development of his protocols for mapping dermatomes by analyzing his lectures and publications from 1908 to 1939, focusing on his Schorstein Memorial Lecture in 1932 and his use of the isolation (Sherrington) method, in which a single dorsal root is spared in a sequence of resections (dorsal rhizotomies). Because of the absence of medical records for Foerster's patients, we also review eyewitness accounts of his operating technique, his occasional comments on patients, and the issue of consent. There appears to be no medical justification-at that time or currently-for Foerster's use of the Sherrington method to map dermatomes L1, L5, S1, and S2, and in our view, these results were obtained unethically. Hence, clinicians and researchers who use his maps should acknowledge those whom Foerster exploited in order to produce them.

A particulate saponin/TLR agonist vaccine adjuvant alters lymph flow and modulates adaptive immunity.

Saponins are potent and safe vaccine adjuvants, but their mechanisms of action remain incompletely understood. Here, we explored the properties of several saponin formulations, including immune-stimulatory complexes (ISCOMs) formed by the self-assembly of saponin and phospholipids in the absence or presence of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA). We found that MPLA self-assembles with saponins to form particles physically resembling ISCOMs, which we termed saponin/MPLA nanoparticles (SMNP). Saponin-containing adjuvants exhibited distinctive mechanisms of action, altering lymph flow in a mast cell­dependent manner and promoting antigen entry into draining lymph nodes. SMNP was particularly effective, exhibiting even greater potency than the compositionally related adjuvant AS01B in mice, and primed robust germinal center B cell, TFH, and HIV tier 2 neutralizing antibodies in nonhuman primates. Together, these findings shed new light on mechanisms by which saponin adjuvants act to promote the immune response and suggest that SMNP may be a promising adjuvant in the setting of HIV, SARS-CoV-2, and other pathogens.