Proteins required for stereocilia elongation during mammalian hair cell development ensure precise and steady heights during adult life.

The hair bundle, or stereocilia bundle, is the mechanosensory compartment of hair cells (HCs) in the inner ear. To date, most mechanistic studies have focused on stereocilia bundle morphogenesis, and it remains unclear how this organelle critical for hearing preserves its precise dimensions during life in mammals. The GPSM2-GNAI complex occupies the distal tip of stereocilia in the tallest row and is required for their elongation during development. Here, we ablate GPSM2-GNAI in adult mouse HCs after normal stereocilia elongation is completed. We observe a progressive height reduction of the tallest row stereocilia totaling ~600 nm after 12 wk in Gpsm2 mutant inner HCs. To measure GPSM2 longevity at tips, we generated a HaloTag-Gpsm2 mouse strain and performed pulse-chase experiments in vivo. Estimates using pulse-chase or tracking loss of GPSM2 immunolabeling following Gpsm2 inactivation suggest that GPSM2 is relatively long-lived at stereocilia tips with a half-life of 9 to 10 d. Height reduction coincides with dampened auditory brainstem responses evoked by low-frequency stimuli in particular. Finally, GPSM2 is required for normal tip enrichment of elongation complex (EC) partners MYO15A, WHRN, and EPS8, mirroring their established codependence during development. Taken together, our results show that the EC is also essential in mature HCs to ensure precise and stable stereocilia height and for sensitive detection of a full range of sound frequencies.

IMPACT OF REAL-LIFE ENVIRONMENTAL EXPOSURES ON REPRODUCTION: Evidence for reproductive health effects following exposure to hydraulic fracturing chemical mixtures.

In Brief: Unconventional oil and natural gas (UOG) operations, particularly hydraulic fracturing, have revolutionized oil and gas production, using and containing complex mixtures of chemicals that may impact reproductive health. While there is growing evidence for effects on births in hydraulic fracturing/UOG regions and good mechanistic evidence for potential reproductive toxicity, there is much research still needed to make firm conclusions about these practices and reproductive health. Abstract: Unconventional oil and natural gas (UOG) operations have emerged over the last four decades to transform oil and gas production in the United States and globally by unlocking previously inaccessible hydrocarbon deposits. UOG development utilizes many compounds associated with conventional oil and gas, as well as some specific to UOG extraction, particularly during hydraulic fracturing (HF). While research is increasing on UOG chemicals and their mixtures, this review discusses the current evidence for reproductive toxicity following exposures to UOG/HF mixtures. These complex chemical mixtures have been demonstrated to interact with numerous mechanisms known to influence reproductive health. A growing number of environmental and controlled laboratory testing studies have reported adverse reproductive health effects in animals exposed to various UOG chemical mixtures. An expanding body of epidemiological literature has assessed adverse birth outcomes, although none has directly examined reproductive measures such as time to pregnancy, semen quality, and other direct measures of fertility. The existing literature provides moderate evidence for decreased birth weights, increased risk of small for gestational age and/or preterm birth, increased congenital abnormalities, and increased infant mortality, though importantly, studies are widely variable in methods used. Most studies utilized distance from UOG operations as an exposure proxy and did not measure actual chemical exposures experienced by those living near these operations. As such, while there is growing evidence for effects on births in these regions and good mechanistic evidence for potential reproductive toxicity, there is much research still needed to make firm conclusions about UOG development and reproductive health.

Altered oxidative stress and antioxidant biomarkers concentrations in pregnant individuals exposed to oil and gas sites in Northeastern British Columbia.

Northeastern British Columbia is a region of prolific unconventional oil and gas (UOG) activity. UOG activity can release volatile organic compounds (VOCs) which can elevate oxidative stress and disrupt antioxidant activity in exposed pregnant individuals, potentially increasing the risk of adverse pregnancy outcomes. This study measured biomarkers of oxidative stress and antioxidant activity in pooled urine samples of 85 pregnant individuals living in Northeastern British Columbia, to analyze associations between indoor air VOCs, oil and gas well density and proximity metrics, and biomarker concentrations. Concentrations of catalase, superoxide dismutase (SOD), glutathione S-transferase, total antioxidant capacity, 6-hydroxymelatonin sulfate (aMT6s), malondialdehyde (MDA), 8-hydroxy-2'-deoxyguanosine (8-OHdG), and 8-isoprostane were measured using assay kits. Associations between exposure metrics and biomarker concentrations were determined using multiple linear regression models adjusted for biomarker-specific covariables. UOG proximity was associated with decreased SOD and 8-OHdG. Decreased 8-OHdG was associated with increased proximity to all wells. Decreased aMT6s were observed with increased indoor air hexanal concentrations. MDA was negatively associated with indoor air 1,4-dioxane concentrations. No statistically significant associations were found between other biomarkers and exposure metrics. Although some associations linked oil and gas activity to altered oxidative stress and antioxidant activity, the possibility of chance findings due to the large number of tests cannot be discounted. This study shows that living near UOG wells may alter oxidative stress and antioxidant activity in pregnant individuals. More research is needed to elucidate underlying mechanisms and to what degree UOG activity affects oxidative stress and antioxidant activity.

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis.

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3, and GNAO proteins, but may also induce unrelated defects. Here, we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner G protein signaling modulator 2 (GPSM2), whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the sub-cellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: (1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and (2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

TopBP1 utilises a bipartite GINS binding mode to support genome replication.

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1. Mutation analysis reveals that either surface is individually able to support TopBP1-GINS interaction, albeit with reduced affinity. Consistently, either surface is sufficient for replication origin firing in Xenopus egg extracts and becomes essential in the absence of the other. The TopBP1-GINS interaction appears sterically incompatible with simultaneous binding of DNA polymerase epsilon (Polε) to GINS when bound to Mcm2-7-Cdc45, although TopBP1-BRCT4 and the Polε subunit PolE2 show only partial competitivity in binding to Psf1. Our TopBP1-GINS model improves the understanding of the recently characterised metazoan pre-loading complex. It further predicts the coordination of three molecular origin firing processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

Improved survival in COVID-19 related sepsis and ARDS treated with a unique "triple therapy" including therapeutic plasma exchange: A single center retrospective analysis.

BACKGROUND: Throughout the COVID-19 pandemic, the mortality of critically ill patients remained high. Our group developed a treatment regimen targeting sepsis and ARDS which we labeled "triple therapy" consisting of (1) corticosteroids, (2) therapeutic plasma exchange (TPE), and (3) timely intubation with lung protective ventilation. Our propensity analysis assesses the impact of triple therapy on survival in COVID-19 patients with sepsis and ARDS. METHODS: Retrospective propensity analysis comparing triple therapy to no triple therapy in adult critically ill COVID-19 patients admitted to the Intensive Care Unit at Lexington Medical Center from 1 March 2020 through 31 October 2021. RESULTS: Eight hundred and fifty-one patients were admitted with COVID-19 and 53 clinical and laboratory variables were analyzed. Multivariable analysis revealed that triple therapy was associated with increased survival (OR: 1.91; P = .008). Two propensity score-adjusted models demonstrated an increased likelihood of survival in patients receiving triple therapy. Patients with thrombocytopenia were among those most likely to experience increased survival if they received early triple therapy. Decreased survival was observed with endotracheal intubation ≥7 days from hospital admission (P < .001) and there was a trend toward decreased survival if TPE was initiated ≥6 days from hospital admission (P = .091). CONCLUSION: Our analysis shows that early triple therapy, defined as high-dose methylprednisolone, TPE, and timely invasive mechanical ventilation within the first 96 hours of admission, may improve survival in critically ill septic patients with ARDS secondary to COVID-19 infection. Further studies are needed to define specific phenotypes and characteristics that will identify those patients most likely to benefit.

Introducing a framework to support the identification and tackling of health inequalities within specialised services.

BACKGROUND: The potential for addressing healthcare inequalities in prescribed specialised services has historically been overlooked. There is evidence that prescribed specialised services can exacerbate inequalities even though they are often accessed at the end of complex pathways and by relatively small numbers of people. Leadership is required to facilitate a systematic approach to identifying and addressing inequalities in this area. METHODS: A rapid literature review of articles from 2015 onwards and engagement with stakeholders was used to inform the development of a framework that both supports the identification of health inequalities within specialised services and provides recommendations for how to address them. RESULTS: The framework aligns with existing national approaches in England to addressing health inequalities in other healthcare settings. It is prepopulated with features of services that may create inequalities and recommended ways of addressing them and can be readily adapted to suit population specific needs. CONCLUSION: The potential for addressing health inequalities should be considered at all points along a healthcare pathway. Local service leaders need to be empowered and encouraged to identify and deliver on opportunities for change to continually improve patient access, experience and outcomes.

Inhibitory G proteins play multiple roles to polarize sensory hair cell morphogenesis.

Inhibitory G alpha (GNAI or Gαi) proteins are critical for the polarized morphogenesis of sensory hair cells and for hearing. The extent and nature of their actual contributions remains unclear, however, as previous studies did not investigate all GNAI proteins and included non-physiological approaches. Pertussis toxin can downregulate functionally redundant GNAI1, GNAI2, GNAI3 and GNAO proteins, but may also induce unrelated defects. Here we directly and systematically determine the role(s) of each individual GNAI protein in mouse auditory hair cells. GNAI2 and GNAI3 are similarly polarized at the hair cell apex with their binding partner GPSM2, whereas GNAI1 and GNAO are not detected. In Gnai3 mutants, GNAI2 progressively fails to fully occupy the subcellular compartments where GNAI3 is missing. In contrast, GNAI3 can fully compensate for the loss of GNAI2 and is essential for hair bundle morphogenesis and auditory function. Simultaneous inactivation of Gnai2 and Gnai3 recapitulates for the first time two distinct types of defects only observed so far with pertussis toxin: 1) a delay or failure of the basal body to migrate off-center in prospective hair cells, and 2) a reversal in the orientation of some hair cell types. We conclude that GNAI proteins are critical for hair cells to break planar symmetry and to orient properly before GNAI2/3 regulate hair bundle morphogenesis with GPSM2.

RGS12 polarizes the GPSM2-GNAI complex to organize and elongate stereocilia in sensory hair cells.

Inhibitory G proteins (GNAI/Gαi) bind to the scaffold G protein signaling modulator 2 (GPSM2) to form a conserved polarity complex that regulates cytoskeleton organization. GPSM2 keeps GNAI in a guanosine diphosphate (GDP)-bound state, but how GPSM2-GNAI is generated or relates to heterotrimeric G protein signaling remains unclear. We find that RGS12, a GTPase-activating protein (GAP), is required to polarize GPSM2-GNAI at the hair cell apical membrane and to organize mechanosensory stereocilia in rows of graded heights. Accordingly, RGS12 and the guanine nucleotide exchange factor (GEF) DAPLE are asymmetrically co-enriched at the hair cell apical junction, and Rgs12 mouse mutants are deaf. GPSM2 and RGS12 share GoLoco motifs that stabilize GNAI(GDP), and GPSM2 outcompetes RGS12 to bind GNAI. Our results suggest that polarized GEF/GAP junctional activity might dissociate heterotrimeric G proteins, generating free GNAI(GDP) for GPSM2 at the adjacent apical membrane. GPSM2-GNAI(GDP), in turn, imparts asymmetry to the forming stereocilia to enable sensory function in hair cells.

Structure of the human RAD17-RFC clamp loader and 9-1-1 checkpoint clamp bound to a dsDNA-ssDNA junction.

The RAD9-RAD1-HUS1 (9-1-1) clamp forms one half of the DNA damage checkpoint system that signals the presence of substantial regions of single-stranded DNA arising from replication fork collapse or resection of DNA double strand breaks. Loaded at the 5'-recessed end of a dsDNA-ssDNA junction by the RAD17-RFC clamp loader complex, the phosphorylated C-terminal tail of the RAD9 subunit of 9-1-1 engages with the mediator scaffold TOPBP1 which in turn activates the ATR kinase, localised through the interaction of its constitutive partner ATRIP with RPA-coated ssDNA. Using cryogenic electron microscopy (cryoEM) we have determined the structure of a complex of the human RAD17-RFC clamp loader bound to human 9-1-1, engaged with a dsDNA-ssDNA junction. The structure answers the key questions of how RAD17 confers specificity for 9-1-1 over PCNA, and how the clamp loader specifically recognises the recessed 5' DNA end and fixes the orientation of 9-1-1 on the ssDNA.

Coherent Interactions between Silicon-Vacancy Centers in Diamond.

We report tunable excitation-induced dipole-dipole interactions between silicon-vacancy color centers in diamond at cryogenic temperatures. These interactions couple centers into collective states, and excitation-induced shifts tag the excitation level of these collective states against the background of excited single centers. By characterizing the phase and amplitude of the spectrally resolved interaction-induced signal, we observe oscillations in the interaction strength and population state of the collective states as a function of excitation pulse area. Our results demonstrate that excitation-induced dipole-dipole interactions between color centers provide a route to manipulating collective intercenter states in the context of a congested, inhomogeneous ensemble.

High-Frequency Oscillatory Ventilation for Refractory Hypoxemia in Severe COVID-19 Pneumonia: A Small Case Series.

BACKGROUND COVID-19 continues to place a tremendous burden on the healthcare system, with most deaths resulting from respiratory failure. Management strategies have varied, but the mortality rate for mechanically ventilated patients remains high. Conventional management with ARDSnet ventilation can improve outcomes but alternative and adjunct treatments continue to be explored. High-frequency oscillatory ventilation (HFOV), a modality now rarely used in adult critical care medicine, may offer an alternative treatment option by maximizing lung protection and limiting oxygen toxicity in critically ill patients failing conventional ventilator strategies. CASE REPORT We present 3 patients with severe acute respiratory distress syndrome (ARDS) and sepsis due to COVID-19 who all improved clinically after transitioning from conventional ventilation to HFOV. Two patients developed refractory hypoxemia with hemodynamic instability and multiple organ failure requiring vasopressor support and renal replacement therapy. After failing to improve with all available therapies, both patients stabilized and ultimately improved after being placed on HFOV. The third patient developed severe volutrauma/barotrauma despite extreme lung protection and ARDSnet ventilation. He showed improvement in oxygenation and signs of lung trauma slowly improved after initiating HFOV. All 3 patients were ultimately liberated from mechanical ventilation and discharged from the hospital to return to functional independence. CONCLUSIONS Our experience suggests that HFOV offers advantages in the management of certain critically ill patients with ARDS due to COVID-19 pneumonia and might be considered in cases refractory to standard management strategies.

Early career researchers benefit from inclusive, diverse and international collaborations: Changing how academic institutions utilize the seminar series.

Efforts to make research environments more inclusive and diverse are beneficial for the next generation of Great Lakes researchers. The global COVID-19 pandemic introduced circumstances that forced graduate programs and academic institutions to re-evaluate and promptly pivot research traditions, such as weekly seminar series, which are critical training grounds and networking opportunities for early career researchers (ECRs). While several studies have established that academics with funded grants and robust networks were better able to weather the abrupt changes in research and closures of institutions, ECRs did not. In response, both existing and novel partnerships provided a resilient network to support ECRs at an essential stage of their career development. Considering these challenges, we sought to re-frame the seminar series as a virtual collaboration for ECRs. Two interdisciplinary graduate programs, located in different countries (Windsor, Canada, and Detroit, USA) invested in a year-long partnership to deliver a virtual-only seminar series that intentionally promoted: the co-creation of protocols and co-led roles, the amplification of justice, equity, diversity and inclusion throughout all aspects of organization and representation, engagement and amplification through social media, the integration of social, scientific and cultural research disciplines, all of which collectively showcased the capacity of our ECRs to lead, organize and communicate. This approach has great potential for application across different communities to learn through collaboration and sharing, and to empower the next generation to find new ways of working together.

Phosphorylation-dependent assembly of DNA damage response systems and the central roles of TOPBP1.

The cellular response to DNA damage (DDR) that causes replication collapse and/or DNA double strand breaks, is characterised by a massive change in the post-translational modifications (PTM) of hundreds of proteins involved in the detection and repair of DNA damage, and the communication of the state of damage to the cellular systems that regulate replication and cell division. A substantial proportion of these PTMs involve targeted phosphorylation, which among other effects, promotes the formation of multiprotein complexes through the specific binding of phosphorylated motifs on one protein, by specialised domains on other proteins. Understanding the nature of these phosphorylation mediated interactions allows definition of the pathways and networks that coordinate the DDR, and helps identify new targets for therapeutic intervention that may be of benefit in the treatment of cancer, where DDR plays a key role. In this review we summarise the present understanding of how phosphorylated motifs are recognised by BRCT domains, which occur in many DDR proteins. We particularly focus on TOPBP1 - a multi-BRCT domain scaffold protein with essential roles in replication and the repair and signalling of DNA damage.

CK2 Phosphorylation of Human Papillomavirus 16 E2 on Serine 23 Promotes Interaction with TopBP1 and Is Critical for E2 Interaction with Mitotic Chromatin and the Viral Life Cycle.

During the human papillomavirus 16 (HPV16) life cycle, the E2 protein interacts with host factors to regulate viral transcription, replication, and genome segregation/retention. Our understanding of host partner proteins and their roles in E2 functions remains incomplete. Here we demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 in vitro and in vivo and that E2 is phosphorylated on this residue during the HPV16 life cycle. We investigated the consequences of mutating serine 23 on E2 functions. E2-S23A (E2 with serine 23 mutated to alanine) activates and represses transcription identically to E2-WT (wild-type E2), and E2-S23A is as efficient as E2-WT in transient replication assays. However, E2-S23A has compromised interaction with mitotic chromatin compared with E2-WT. In E2-WT cells, both E2 and TopBP1 levels increase during mitosis compared with vector control cells. In E2-S23A cells, neither E2 nor TopBP1 levels increase during mitosis. Introduction of the S23A mutation into the HPV16 genome resulted in delayed immortalization of human foreskin keratinocytes (HFK) and higher episomal viral genome copy number in resulting established HFK. Remarkably, S23A cells had a disrupted viral life cycle in organotypic raft cultures, with a loss of E2 expression and a failure of viral replication. Overall, our results demonstrate that CK2 phosphorylation of E2 on serine 23 promotes interaction with TopBP1 and that this interaction is critical for the viral life cycle. IMPORTANCE Human papillomaviruses are causative agents in around 5% of all cancers, with no specific antiviral therapeutics available for treating infections or resultant cancers. In this report, we demonstrate that phosphorylation of HPV16 E2 by CK2 promotes formation of a complex with the cellular protein TopBP1 in vitro and in vivo. This complex results in stabilization of E2 during mitosis. We demonstrate that CK2 phosphorylates E2 on serine 23 in vivo and that CK2 inhibitors disrupt the E2-TopBP1 complex. Mutation of E2 serine 23 to alanine disrupts the HPV16 life cycle, hindering immortalization and disrupting the viral life cycle, demonstrating a critical function for this residue.

Hidden Silicon-Vacancy Centers in Diamond.

We characterize a high-density sample of negatively charged silicon-vacancy (SiV^{-}) centers in diamond using collinear optical multidimensional coherent spectroscopy. By comparing the results of complementary signal detection schemes, we identify a hidden population of SiV^{-} centers that is not typically observed in photoluminescence and which exhibits significant spectral inhomogeneity and extended electronic T_{2} times. The phenomenon is likely caused by strain, indicating a potential mechanism for controlling electric coherence in color-center-based quantum devices.

EMX2-GPR156-Gαi reverses hair cell orientation in mechanosensory epithelia.

Hair cells detect sound, head position or water movements when their mechanosensory hair bundle is deflected. Each hair bundle has an asymmetric architecture that restricts stimulus detection to a single axis. Coordinated hair cell orientations within sensory epithelia further tune stimulus detection at the organ level. Here, we identify GPR156, an orphan GPCR of unknown function, as a critical regulator of hair cell orientation. We demonstrate that the transcription factor EMX2 polarizes GPR156 distribution, enabling it to signal through Gαi and trigger a 180° reversal in hair cell orientation. GPR156-Gαi mediated reversal is essential to establish hair cells with mirror-image orientations in mouse otolith organs in the vestibular system and in zebrafish lateral line. Remarkably, GPR156-Gαi also instructs hair cell reversal in the auditory epithelium, despite a lack of mirror-image organization. Overall, our work demonstrates that conserved GPR156-Gαi signaling is integral to the framework that builds directional responses into mechanosensory epithelia.

Structural basis for recruitment of the CHK1 DNA damage kinase by the CLASPIN scaffold protein.

CHK1 is a protein kinase that functions downstream of activated ATR to phosphorylate multiple targets as part of intra-S and G2/M DNA damage checkpoints. Its role in allowing cells to survive replicative stress has made it an important target for anti-cancer drug discovery. Activation of CHK1 by ATR depends on their mutual interaction with CLASPIN, a natively unstructured protein that interacts with CHK1 through a cluster of phosphorylation sites in its C-terminal half. We have now determined the crystal structure of the kinase domain of CHK1 bound to a high-affinity motif from CLASPIN. Our data show that CLASPIN engages a conserved site on CHK1 adjacent to the substrate-binding cleft, involved in phosphate sensing in other kinases. The CLASPIN motif is not phosphorylated by CHK1, nor does it affect phosphorylation of a CDC25 substrate peptide, suggesting that it functions purely as a scaffold for CHK1 activation by ATR.

Radio frequency polarization modulation based on an optical frequency comb.

We propose a method to generate stabilized radio-frequency polarization modulation based on optical frequency combs. Two pulse trains with the same repetition rate and different offset frequencies generate arbitrary polarization states that are modulated at the offset frequency difference. Long-term stability of the polarization modulation is demonstrated with the modulation frequency at frep/2. Modulation at frep/4 is also demonstrated to show the flexibility of the technique. We employ an electrical delay line to fine-tune the polarization states that constitute the time-dependent modulation.