Oxygen toxicity causes cyclic damage by destabilizing specific Fe-S cluster-containing protein complexes.

Oxygen is toxic across all three domains of life. Yet, the underlying molecular mechanisms remain largely unknown. Here, we systematically investigate the major cellular pathways affected by excess molecular oxygen. We find that hyperoxia destabilizes a specific subset of Fe-S cluster (ISC)-containing proteins, resulting in impaired diphthamide synthesis, purine metabolism, nucleotide excision repair, and electron transport chain (ETC) function. Our findings translate to primary human lung cells and a mouse model of pulmonary oxygen toxicity. We demonstrate that the ETC is the most vulnerable to damage, resulting in decreased mitochondrial oxygen consumption. This leads to further tissue hyperoxia and cyclic damage of the additional ISC-containing pathways. In support of this model, primary ETC dysfunction in the Ndufs4 KO mouse model causes lung tissue hyperoxia and dramatically increases sensitivity to hyperoxia-mediated ISC damage. This work has important implications for hyperoxia pathologies, including bronchopulmonary dysplasia, ischemia-reperfusion injury, aging, and mitochondrial disorders.

Safety outcomes and improvement in pain scores after radiologist-performed fluoroscopy-guided interlaminar cervical epidural steroid injection.

INTRODUCTION: Image-guided spine injections are an important tool in the management of patients with a variety of spinal pathologies. Our practice offers radiologist-performed fluoroscopy-guided interlaminar cervical epidural steroid injection (ESI) routinely performed in the outpatient setting. The purpose of this study was to evaluate clinical outcomes and improvement in pain scores after radiologist-performed cervical ESI. METHODS: An institutional database was used to retrospectively identify cervical injections performed between October 2016 and October 2022. All injections were performed at the C7-T1 level utilizing 1.0 mL of 10 mg/mL dexamethasone without epidural anesthetic. The Numerical Rating Scale (NRS) was used to assess pain improvement. Cervical MRI was reviewed to assess pre-injection cervical disease severity. Patient charts were reviewed for any post-injection complications. RESULTS: A total of 251 cervical injections in 186 patients met our inclusion criteria with mean clinical follow up of 28.5 months (range 0.2 - 73.0 months). No patients experienced any major complications after injection. Post-injection pain scores were available for 218 of 251 injections (86.9%) with mean follow-up of 11.8 days (range 6 - 57 days). There was a significant improvement in pain scores from a mean pre-injection NRS score of 5.2/10 to 3.0/10 (p < .0001). 117 patients (53.7%) reported ≥ 50% improvement after injection. Patients who had prior injection were more likely to report ≥ 50% pain improvement after subsequent injection (p = .012). CONCLUSION: Radiologist-performed fluoroscopy-guided interlaminar cervical ESI at the C7-T1 level is a safe and effective tool in the management of patients with cervical pathology.

E3 ubiquitin ligase FBXW7 balances airway cell fates.

The airway epithelium is composed of multiple cell types each with designated roles. A stereotyped ratio of these cells is essential for proper airway function. Imbalance of airway cell types underlies many lung diseases, including chronic obstructive pulmonary disease (COPD) and asthma. While a number of signals and transcription factors have been implicated in airway cell specification, how cell numbers are coordinated, especially at the protein level is poorly understood. Here we show that in the mouse trachea which contain epithelial cell types similar to human airway, epithelium-specific inactivation of Fbxw7, which encodes an E3 ubiquitin ligase, led to reduced club and ciliated cells, increased goblet cells, and ectopic P63-negative, Keratin5-positive transitory basal cells in the luminal layer. The protein levels of FBXW7 targets including NOTCH1, KLF5 and TGIF were increased. Inactivation of either Notch1, Klf5 but not Tgif genes in the mutant background led to attenuation of selected aspects of the phenotypes, suggesting that FBXW7 acts through different targets to control different cell fates. These findings demonstrate that protein-level regulation by the ubiquitin proteasome system is critical for balancing airway cell fates.

PIN and CCCH Zn-finger domains coordinate RNA targeting in ZC3H12 family endoribonucleases.

The CCCH-type zinc finger (ZnF) containing ZC3H12 ribonucleases are crucial in post-transcriptional immune homoeostasis with ZC3H12A being the only structurally studied member of the family. In this study, we present a structural-biochemical characterization of ZC3H12C, which is linked with chronic immune disorders like psoriasis. We established that the RNA substrate is cooperatively recognized by the PIN and ZnF domains of ZC3H12C and analyzed the crystal structure of ZC3H12C bound to a single-stranded RNA substrate. The RNA engages in hydrogen-bonded contacts and stacking interactions with the PIN and ZnF domains simultaneously. The ZC3H12 ZnF shows unprecedented structural features not previously observed in any member of the CCCH-ZnF family and utilizes stacking interactions via a unique combination of spatially conserved aromatic residues to align the target transcript in a bent conformation onto the ZnF scaffold. Further comparative structural analysis of ZC3H12 CCCH-ZnF suggests that a trinucleotide sequence is recognized by ZC3H12 ZnF in target RNA. Our work not only describes the initial structure-biochemical study on ZC3H12C, but also provides the first molecular insight into RNA recognition by a ZC3H12 family member. Finally, our work points to an evolutionary code for RNA recognition adopted by CCCH-type ZnF proteins.

E3 ubiquitin ligase MDM2 acts through p53 to control respiratory progenitor cell number and lung size.

The respiratory lineage initiates from the specification of NKX2-1+ progenitor cells that ultimately give rise to a vast gas-exchange surface area. How the size of the progenitor pool is determined and whether this directly impacts final lung size remains poorly understood. Here, we show that epithelium-specific inactivation of Mdm2, which encodes an E3 ubiquitin ligase, led to lethality at birth with a striking reduction of lung size to a single vestigial lobe. Intriguingly, this lobe was patterned and contained all the appropriate epithelial cell types. The reduction of size can be traced to the progenitor stage, when p53, a principal MDM2 protein degradation target, was transiently upregulated. This was followed by a brief increase of apoptosis. Inactivation of the p53 gene in the Mdm2 mutant background effectively reversed the lung size phenotype, allowing survival at birth. Together, these findings demonstrate that p53 protein turnover by MDM2 is essential for the survival of respiratory progenitors. Unlike in the liver, in which genetic reduction of progenitors triggered compensation, in the lung, respiratory progenitor number is a key determinant factor for final lung size.

Essential medicines for cardiovascular diseases in India: Rapid appraisal of policies and processes at the subnational level.

Background The burden of cardiovascular diseases (CVDs) and response to health systems vary widely at the subnational level in India. Our study aimed to assess the variation in state-level access to medicines for CVDs by comparing the essential medicines lists (EMLs) at the national and subnational levels in India and by rapid appraisal of the existing policies and processes of drug procurement. Methods We assessed the inclusion of six classes of medicines for CVDs in the recent and publicly available national and subnational EMLs from July to September 2018 in the states of Telangana and Madhya Pradesh. We examined the drug procurement and distribution policies and processes using documentary review and five key informant interviews between March and June 2018. Results The WHO's EML, India's national EML, and 21 of 28 publicly available (75%) Indian state and Union Territory EMLs included all six classes of essential medicines for CVDs. However, some medicines were not included in the policy packages of essential medicines meant for primary health centres. Both the states used centralized tendering and decentralized distribution as part of the public sector drug procurement process. The requirement was based on the previous year's consumption. The approximate time between procurement planning and distribution was 7-8 months in both the states. Conclusion Substantial variation exists in the selection of drugs for CVDs in EMLs at the subnational level in India. Improving forecasting techniques for requirement of medicines and reducing time lags between forecasting and distribution to health facilities may allow for better access to essential medicines.

FGF-induced Pea3 transcription factors program the genetic landscape for cell fate determination.

FGF signaling is a potent inducer of lacrimal gland development in the eye, capable of transforming the corneal epithelium into glandular tissues. Here, we show that genetic ablation of the Pea3 family of transcription factors not only disrupted the ductal elongation and branching of the lacrimal gland, but also biased the lacrimal gland epithelium toward an epidermal cell fate. Analysis of high-throughput gene expression and chromatin immunoprecipitation data revealed that the Pea3 genes directly control both the positive and negative feedback loops of FGF signaling. Importantly, Pea3 genes are also required to suppress aberrant Notch signaling which, if gone unchecked, can compromise lacrimal gland development by preventing the expression of both Sox and Six family genes. These results demonstrate that Pea3 genes are key FGF early response transcriptional factors, programing the genetic landscape for cell fate determination.

Magnetic resonance neurography findings in three critically ill COVID-19 patients with new onset of extremity peripheral neuropathy.

PURPOSE: Coronavirus-19 (COVID-19) is most commonly associated with respiratory syndromes, although patients are presenting more frequently with neurological symptoms. When they occur, neurological conditions most commonly involve the central nervous system, and peripheral nervous system effects, particularly in the extremities, have been less commonly described. The mechanisms of peripheral neuropathy in critically ill patients with COVID-19 are likely to be multifactorial, and extremity peripheral nerve imaging in these cases has not been well described. CASE PRESENTATION: In this case series, we describe the magnetic resonance neurography (MRN) findings in 3 critically ill patients who presented with new onset of peripheral neuropathies in the extremities, and we discuss possible common mechanisms of nerve injury, including the role of position-related nerve injury. CONCLUSIONS: MRN can be useful in identifying and localizing peripheral nerve abnormalities in the extremities of COVID-19 patients, and patients who are placed in the prone position during ventilation may be more susceptible to these injuries.

A novel form of RNA double helix based on G·U and C·A+ wobble base pairing.

Wobble base pairs are critical in various physiological functions and have been linked to local structural perturbations in double-helical structures of nucleic acids. We report a 1.38-Å resolution crystal structure of an antiparallel octadecamer RNA double helix in overall A conformation, which includes a unique, central stretch of six consecutive wobble base pairs (W helix) with two G·U and four rare C·A+ wobble pairs. Four adenines within the W helix are N1-protonated and wobble-base-paired with the opposing cytosine through two regular hydrogen bonds. Combined with the two G·U pairs, the C·A+ base pairs facilitate formation of a half turn of W-helical RNA flanked by six regular Watson-Crick base pairs in standard A conformation on either side. RNA melting experiments monitored by differential scanning calorimetry, UV and circular dichroism spectroscopy demonstrate that the RNA octadecamer undergoes a pH-induced structural transition which is consistent with the presence of a duplex with C·A+ base pairs at acidic pH. Our crystal structure provides a first glimpse of an RNA double helix based entirely on wobble base pairs with possible applications in RNA or DNA nanotechnology and pH biosensors.

Alx4 relays sequential FGF signaling to induce lacrimal gland morphogenesis.

The sequential use of signaling pathways is essential for the guidance of pluripotent progenitors into diverse cell fates. Here, we show that Shp2 exclusively mediates FGF but not PDGF signaling in the neural crest to control lacrimal gland development. In addition to preventing p53-independent apoptosis and promoting the migration of Sox10-expressing neural crests, Shp2 is also required for expression of the homeodomain transcription factor Alx4, which directly controls Fgf10 expression in the periocular mesenchyme that is necessary for lacrimal gland induction. We show that Alx4 binds an Fgf10 intronic element conserved in terrestrial but not aquatic animals, underlying the evolutionary emergence of the lacrimal gland system in response to an airy environment. Inactivation of ALX4/Alx4 causes lacrimal gland aplasia in both human and mouse. These results reveal a key role of Alx4 in mediating FGF-Shp2-FGF signaling in the neural crest for lacrimal gland development.

Magnetic resonance imaging of the wrist and hand.

The anatomy of the wrist and hand is complex and contains numerous small structures. Magnetic resonance imaging (MRI) is often an ideal imaging modality in the assessment of various traumatic and pathologic conditions of this region, and it is frequently performed after initial radiographs. In this manuscript we describe the normal anatomy, imaging techniques, and MRI findings of various traumatic and pathologic conditions of the wrist and hand including occult fractures, osteonecrosis, ligamentous and tendon injuries, and entrapment neuropathies.

Correction to: Sonographic evaluation of uncommonly assessed upper extremity peripheral nerves: anatomy, technique, and clinical syndromes.

The original version of this article unfortunately contained mistake. Fig. 13a (Anatomy of the Ulnar Digital nerve of the Thumb) as originally published erroneously depicts the ulnar digital nerve of the thumb as a branch of the ulnar nerve.

Initial experience with dual-energy computed tomography-guided bone biopsies of bone lesions that are occult on monoenergetic CT.

OBJECTIVE: Our purpose was to determine whether dual-energy CT (DECT), specifically the bone marrow setting of the virtual noncalcium (VNCa) algorithm, could be used to identify and accurately biopsy suspected bone malignancies that were visible on magnetic resonance imaging (MRI), nuclear bone scintigraphy, or positron-emission tomography/computed tomography (PET/CT), but occult on monoenergetic computed tomography (CT) by virtue of being either isodense or nearly isodense to surrounding normal bone. MATERIALS AND METHODS: We present 4 cases in which DECT was used to detect various malignant bone lesions and was successfully used to direct percutaneous DECT-guided bone biopsies. RESULTS: Two of the lesions were solid tumor metastases (breast and prostate carcinoma), whereas two others were hematological malignancies (leukemia and lymphoma). This technique enabled us to confidently and accurately direct the biopsy needle into the target lesion. CONCLUSION: The authors demonstrate that the DECT VNCa bone marrow algorithm may be helpful in identifying isodense bone lesions of various histologies and may be used to guide percutaneous bone biopsies. This technique may help to maximize diagnostic yield, minimize the number of passes into the region of concern, and prevent patients from undergoing repeat biopsy.

Sonographic evaluation of uncommonly assessed upper extremity peripheral nerves: anatomy, technique, and clinical syndromes.

Targeted ultrasound of the median, ulnar, and radial nerves is a well-established technique for suspected upper extremity peripheral neuropathy. However, sonographic imaging of the brachial plexus and smaller peripheral nerve branches is more technically difficult and the anatomy is less familiar to many radiologists. As imaging techniques improve, many clinicians refer patients for imaging of previously less-familiar structures. In addition, some patients may present with injuries that could involve local neurovascular structures. Finally, patients presenting with isolated peripheral neuropathies may be referred for perineural injections with local anesthetic for diagnostic purposes, or steroid for therapeutic reasons. This requires sonologists to have a firm understanding of the courses of these nerves and the surrounding anatomic landmarks that can be used to accurately identify and characterize them. We discuss clinical syndromes referable to specific peripheral nerve branches in the upper extremity, the relevant anatomy, and sonographic technique.

Optimizing methods to quantify intramuscular fat in rotator cuff tears with normalization.

OBJECTIVE: To determine which normalization method may best account for confounding individual factors, such as age or BMI, when quantifying fat infiltration on MRI in patients with rotator cuff tears, the effects of normalization using three different muscles (teres major; triceps brachii; teres minor) were compared. METHODS: Thirty-seven consecutive patients diagnosed with rotator cuff pathology were included. MRI fat-water sequences were used to quantify rotator cuff intramuscular fat (%fat). Three reference muscles (teres major, triceps, teres minor) were used to derive normalized %fat. Relationships between intramuscular %fat and tear size, age, and BMI in each rotator cuff muscle, before and after normalization, were compared with Fisher transformations (α = 0.05). RESULTS: Normalization with teres major ameliorated confounding relationships of age and BMI on rotator cuff %fat. In contrast, normalization with triceps maintained the confounding relationships between %fat and age in supraspinatus (p = 0.03) and infraspinatus/teres minor (p = 0.028). Normalization with teres minor maintained the confounding relationship between %fat and BMI in subscapularis (p = 0.039). Normalization with teres major best-maintained relationships between tear size and infraspinatus/teres minor %fat (p = 0.021). In contrast, normalization with triceps or teres minor eliminated all significant relationships with tear size. CONCLUSIONS: Results of this pilot study suggest normalization to teres major using MRI-based %fat quantification methods can effectively control for individual factors, such as BMI or age, and may have utility in evaluating and monitoring rotator cuff fat infiltration attributed specifically to a tendon tear.

Lacrimal gland development: From signaling interactions to regenerative medicine.

The lacrimal gland plays a pivotal role in keeping the ocular surface lubricated, and protecting it from environmental exposure and insult. Dysfunction of the lacrimal gland results in deficiency of the aqueous component of the tear film, which can cause dryness of the ocular surface, also known as the aqueous-deficient dry eye disease. Left untreated, this disease can lead to significant morbidity, including frequent eye infections, corneal ulcerations, and vision loss. Current therapies do not treat the underlying deficiency of the lacrimal gland, but merely provide symptomatic relief. To develop more sustainable and physiological therapies, such as in vivo lacrimal gland regeneration or bioengineered lacrimal gland implants, a thorough understanding of lacrimal gland development at the molecular level is of paramount importance. Based on the structural and functional similarities between rodent and human eye development, extensive studies have been undertaken to investigate the signaling and transcriptional mechanisms of lacrimal gland development using mouse as a model system. In this review, we describe the current understanding of the extrinsic signaling interactions and the intrinsic transcriptional network governing lacrimal gland morphogenesis, as well as recent advances in the field of regenerative medicine aimed at treating dry eye disease. Developmental Dynamics 246:970-980, 2017. © 2017 Wiley Periodicals, Inc.

An allelic series at the paired box gene 6 (Pax6) locus reveals the functional specificity of Pax genes.

The advent of the ocular and nervous system in metazoan evolution coincides with the diversification of a single ancestral paired box (Pax) gene into Pax6, Pax6(5a), and Pax2. To investigate the role of these Pax genes in neural development, we have generated an allelic series of knock-in models at the Pax6 locus. We showed that although Pax6(5a) and Pax2 could not replace Pax6 for its autoregulation in lens induction or for neural differentiation in retina, Pax6(5a) was sufficient for corneal-lenticular detachment. In brain development, cell proliferation in the cerebral cortex and dorsoventral patterning of the telencephalon and neural tube were partially rescued in either knock-in mutant. Contrary to the previous belief, our genetic studies showed that the Pax6 isoform Pax6(5a) could potentially play a role in neuronal differentiation in brain development. Importantly, Pax2 showed greater rescue efficiency than Pax6(5a) in the telencephalon even though the latter was identical to Pax6 outside the paired domain. In studying Ngn2, a Pax6 direct target gene in telencephalon, we showed that the level of Ngn2 expression correlated with the in vitro binding of Pax2, Pax6, and Pax6(5a) paired domain on its enhancer. Our results show that Pax6 is uniquely required for eye development, but in brain development, Pax6 can be functionally substituted by related Pax family genes that share a similar paired domain binding specificity.

The structural landscape of Microprocessor Mediated pri-let-7 miRNAs processing.

miRNA biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryo-EM and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has structural plasticity to accommodate different pri-miRNAs. These also revealed key structural features of the 5' UG sequence motif, more comprehensively represented as the "fUN" motif. Our analysis explains how the bulged nucleotide in class-II pri-let-7 members alters Drosha cleavage, generating a noncanonical precursor with 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 interacts with the CNNC motif and Drosha's PAZ-like domain, to promote proper Drosha loading onto the basal hairpin junction. Overall, our work illuminates the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.