A Blueprint for Characterizing Senescence.

Given the heterogeneity of senescent cells, our knowledge of both the drivers and consequences of cellular senescence in tissues and organs remains limited, as is our understanding of how this process could be harnessed for human health. Here we identified five broad areas that would help propel the field forward.

Update on and Future Directions for Use of Anti-SARS-CoV-2 Antibodies: National Institutes of Health Summit on Treatment and Prevention of COVID-19.

As the fourth wave of the SARS-CoV-2 pandemic encircles the globe, there remains an urgent challenge to identify safe and effective treatment and prevention strategies that can be implemented in a range of health care and clinical settings. Substantial advances have been made in the use of anti-SARS-CoV-2 antibodies to mitigate the morbidity and mortality associated with COVID-19. On 15 June 2021, the National Institutes of Health, in collaboration with the U.S. Food and Drug Administration, convened a virtual summit to summarize existing knowledge on anti-SARS-CoV-2 antibodies and to identify key unanswered scientific questions to further catalyze the clinical development and implementation of antibodies.

Evidence of increasing juvenile white sharks' (Carcharodon carcharias) habitat use at the Northern Channel Islands.

Juvenile white sharks (Carcharodon carcharias) typically aggregate along coastal beaches; however, high levels of recruitment and shifting oceanographic conditions may be causing habitat use expansions. Telemetry data indicate increased habitat use at the Northern Channel Islands (California, USA) by juvenile white shark that may be in response to increased population density at aggregation locations, or anomalous oceanographic events that impact habitat use or expand available habitat. Findings illustrate the need for long-term movement monitoring and understanding drivers of habitat use shifts and expansion to improve ecosystem management.

The Influence of Retrograde Femoral Nail Removal With and Without Interpositional Fat Grafting on Distal Femoral Physeal Behavior: A Sheep Study.

BACKGROUND: Previous investigations have demonstrated that up to 7% of the distal femoral physis can be violated using a rigid, retrograde nail without growth inhibition or arrest. The purpose of this investigation was to evaluate the behavior of the distal femoral physis after retrograde femoral nail removal in a sheep model, with and without placement of an interpositional fat graft. METHODS: Retrograde femoral nails were placed in 8 skeletally immature sheep. Implants were removed at 8 weeks, with the residual defects left open (n=4) or filled with autologous fat graft (n=4). Differences in femoral length between surgical versus contralateral control femurs were measured after an additional 3 (n=4) or 5 months (n=4) before sacrifice, and the physis was evaluated histologically. RESULTS: When compared with control limbs, femoral length was significantly shorter in limbs sacrificed at 3 months (mean: 3.9±1.3 mm; range: 2.7 to 5.7 mm) compared with limbs at 5 months (mean: 1.0±0.4 mm; range: 0.4 to 1.2 mm) ( P =0.005). No significant difference in mean shortening was appreciated in limbs without (2.4±1.6 mm) versus with fat grafting (2.5±2.3 mm) ( P =0.94). Histologic analysis revealed no osteoid formation across the physis in sheep sacrificed at 3 months, whereas there was evidence of early osteoid formation across the physis in sheep at 5 months. All specimens demonstrated evidence of an active physes. CONCLUSIONS: Femurs undergoing retrograde implant placement were significantly shorter when compared with control limbs in sheep sacrificed at 3 months, whereas differences were nominal in sheep sacrificed at 5 months after retrograde implant removal, suggesting growth inhibition with nail removal improved with time. Fat grafting across the distal femoral physis did not result in a significant difference in femoral lengths. Histologic evidence at 5 months revealed early development of a bone bridge, emphasizing the importance of follow-up to skeletal maturity in patients treated with retrograde nailing across an open physis. LEVEL OF EVIDENCE: Level IV.

Observations of a species-record deep dive by a central Pacific female scalloped hammerhead shark (Sphyrna lewini).

A female scalloped hammerhead shark (Sphyrna lewini) conducted a species record deep dive to 1240 m in coastal-pelagic waters off Hawaii Island. This extends the deepest known depth range of the species by over 200 m (650 ft) and highlights the question of the extent to which deep-diving activity is mediated by physiological constraints, such as temperature and oxygen availability.

Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit.

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

MARCKS-related protein regulates cytoskeletal organization at cell-cell and cell-substrate contacts in epithelial cells.

Treatment of epithelial cells with interferon-γ and TNF-α (IFN/TNF) results in increased paracellular permeability. To identify relevant proteins mediating barrier disruption, we performed proximity-dependent biotinylation (BioID) of occludin and found that tagging of MARCKS-related protein (MRP; also known as MARCKSL1) increased ∼20-fold following IFN/TNF administration. GFP-MRP was focused at the lateral cell membrane and its overexpression potentiated the physiological response of the tight junction barrier to cytokines. However, deletion of MRP did not abrogate the cytokine responses, suggesting that MRP is not required in the occludin-dependent IFN/TNF response. Instead, our results reveal a key role for MRP in epithelial cells in control of multiple actin-based structures, likely by regulation of integrin signaling. Changes in focal adhesion organization and basal actin stress fibers in MRP-knockout (KO) cells were reminiscent of those seen in FAK-KO cells. In addition, we found alterations in cell-cell interactions in MRP-KO cells associated with increased junctional tension, suggesting that MRP may play a role in focal adhesion-adherens junction cross talk. Together, our results are consistent with a key role for MRP in cytoskeletal organization of cell contacts in epithelial cells.

Relative Citation Ratio (RCR): A New Metric That Uses Citation Rates to Measure Influence at the Article Level.

Despite their recognized limitations, bibliometric assessments of scientific productivity have been widely adopted. We describe here an improved method to quantify the influence of a research article by making novel use of its co-citation network to field-normalize the number of citations it has received. Article citation rates are divided by an expected citation rate that is derived from performance of articles in the same field and benchmarked to a peer comparison group. The resulting Relative Citation Ratio is article level and field independent and provides an alternative to the invalid practice of using journal impact factors to identify influential papers. To illustrate one application of our method, we analyzed 88,835 articles published between 2003 and 2010 and found that the National Institutes of Health awardees who authored those papers occupy relatively stable positions of influence across all disciplines. We demonstrate that the values generated by this method strongly correlate with the opinions of subject matter experts in biomedical research and suggest that the same approach should be generally applicable to articles published in all areas of science. A beta version of iCite, our web tool for calculating Relative Citation Ratios of articles listed in PubMed, is available at https://icite.od.nih.gov.

The projected timing of climate departure from recent variability.

Ecological and societal disruptions by modern climate change are critically determined by the time frame over which climates shift beyond historical analogues. Here we present a new index of the year when the projected mean climate of a given location moves to a state continuously outside the bounds of historical variability under alternative greenhouse gas emissions scenarios. Using 1860 to 2005 as the historical period, this index has a global mean of 2069 (±18 years s.d.) for near-surface air temperature under an emissions stabilization scenario and 2047 (±14 years s.d.) under a 'business-as-usual' scenario. Unprecedented climates will occur earliest in the tropics and among low-income countries, highlighting the vulnerability of global biodiversity and the limited governmental capacity to respond to the impacts of climate change. Our findings shed light on the urgency of mitigating greenhouse gas emissions if climates potentially harmful to biodiversity and society are to be prevented.

Combining abundance and performance data reveals how temperature regulates coastal occurrences and activity of a roaming apex predator.

The redistribution of species has emerged as one of the most pervasive impacts of anthropogenic climate warming, and presents many societal challenges. Understanding how temperature regulates species distributions is particularly important for mobile marine fauna such as sharks given their seemingly rapid responses to warming, and the socio-political implications of human encounters with some dangerous species. The predictability of species distributions can potentially be improved by accounting for temperature's influence on performance, an elusive relationship for most large animals. We combined multi-decadal catch data and bio-logging to show that coastal abundance and swimming performance of tiger sharks Galeocerdo cuvier are both highest at ~22°C, suggesting thermal constraints on performance may regulate this species' distribution. Tiger sharks are responsible for a large proportion of shark bites on humans, and a focus of controversial control measures in several countries. The combination of distribution and performance data moves towards a mechanistic understanding of tiger shark's thermal niche, and delivers a simple yet powerful indicator for predicting the location and timing of their occurrences throughout coastlines. For example, tiger sharks are mostly caught at Australia's popular New South Wales beaches (i.e. near Sydney) in the warmest months, but our data suggest similar abundances will occur in winter and summer if annual sea surface temperatures increase by a further 1-2°C.

Liver kinase B1 regulates hepatocellular tight junction distribution and function in vivo.

UNLABELLED: Liver kinase B1 (LKB1) and its downstream effector AMP-activated protein kinase (AMPK) play critical roles in polarity establishment by regulating membrane trafficking and energy metabolism. In collagen sandwich-cultured hepatocytes, loss of LKB1 or AMPK impaired apical ABCB11 (Bsep) trafficking and bile canalicular formation. In the present study, we used liver-specific (albumin-Cre) LKB1 knockout mice (LKB1(-/-) ) to investigate the role of LKB1 in the maintenance of functional tight junction (TJ) in vivo. Transmission electron microscopy examination revealed that hepatocyte apical membrane with microvilli substantially extended into the basolateral domain of LKB1(-/-) livers. Immunofluorescence studies revealed that loss of LKB1 led to longer and wider canalicular structures correlating with mislocalization of the junctional protein, cingulin. To test junctional function, we used intravital microscopy to quantify the transport kinetics of 6-carboxyfluorescein diacetate (6-CFDA), which is processed in hepatocytes into its fluorescent derivative 6-carboxyfluorescein (6-CF) and secreted into the canaliculi. In LKB1(-/-) mice, 6-CF remained largely in hepatocytes, canalicular secretion was delayed, and 6-CF appeared in the blood. To test whether 6-CF was transported through permeable TJ, we intravenously injected low molecular weight (3 kDa) dextran in combination with 6-CFDA. In wild-type mice, 3 kDa dextran remained in the vasculature, whereas it rapidly appeared in the abnormal bile canaliculi in LKB1(-/-) mice, confirming that junctional disruption resulted in paracellular exchange between the blood stream and the bile canaliculus. CONCLUSION: LKB1 plays a critical role in regulating the maintenance of TJ and paracellular permeability, which may explain how various drugs, chemicals, and metabolic states that inhibit the LKB1/AMPK pathway result in cholestasis. (Hepatology 2016;64:1317-1329).

Architecture of tight junctions and principles of molecular composition.

The tight junction creates an intercellular barrier limiting paracellular movement of solutes and material across epithelia. Currently many proteins have been identified as components of the tight junction and understanding their architectural organization and interactions is critical to understanding the biology of the barrier. In general the architecture can be conceptualized into compartments with the transmembrane barrier proteins (claudins, occludin, JAM-A, etc.), linked to peripheral scaffolding proteins (such as ZO-1, afadin, MAGI1, etc.) which are in turned linked to actin and microtubules through numerous linkers (cingulin, myosins, protein 4.1, etc.). Within this complex network are associated many signaling proteins that affect the barrier and broader cell functions. The PDZ domain is a commonly used motif to specifically link individual junction protein pairs. Here we review some of the key proteins defining the tight junction and general themes of their organization with the perspective that much will be learned about function by characterizing the detailed architecture and subcompartments within the junction.

Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First PDZ Domain and Claudins.

The molecular seal between epithelial cells, called the tight junction (TJ), is built by several membrane proteins, with claudins playing the most prominent role. The scaffold proteins of the zonula occludens family are required for the correct localization of claudins and hence formation of the TJ. The intracellular C terminus of claudins binds to the N-terminal PDZ domain of zonula occludens proteins (PDZ1). Of the 23 identified human claudin proteins, nine possess a tyrosine at the -6 position. Here we show that the claudin affinity for PDZ1 is dependent on the presence or absence of this tyrosine and that the affinity is reduced if the tyrosine is modified by phosphorylation. The PDZ1 ß2-ß3 loop undergoes a significant conformational change to accommodate this tyrosine. Cell culture experiments support a regulatory role for this tyrosine. Plasticity has been recognized as a critical property of TJs that allow cell remodeling and migration. Our work provides a molecular framework for how TJ plasticity may be regulated.

Biotin ligase tagging identifies proteins proximal to E-cadherin, including lipoma preferred partner, a regulator of epithelial cell-cell and cell-substrate adhesion.

Known proteins associated with the cell-adhesion protein E-cadherin include catenins and proteins involved in signaling, trafficking and actin organization. However, the list of identified adherens junction proteins is likely to be incomplete, limiting investigation into this essential cell structure. To expand the inventory of potentially relevant proteins, we expressed E-cadherin fused to biotin ligase in MDCK epithelial cells, and identified by mass spectrometry neighboring proteins that were biotinylated. The most abundant of the 303 proteins identified were catenins and nearly 40 others that had been previously reported to influence cadherin function. Many others could be rationalized as novel candidates for regulating the adherens junction, cytoskeleton, trafficking or signaling. We further characterized lipoma preferred partner (LPP), which is present at both cell contacts and focal adhesions. Knockdown of LPP demonstrated its requirement for E-cadherin-dependent adhesion and suggested that it plays a role in coordination of the cell-cell and cell-substrate cytoskeletal interactions. The analysis of LPP function demonstrates proof of principle that the proteomic analysis of E-cadherin proximal proteins expands the inventory of components and tools for understanding the function of E-cadherin.

An in vivo analysis of Miromesh--a novel porcine liver prosthetic created by perfusion decellularization.

BACKGROUND: Bioprosthetics derived from human or porcine dermis and intestinal submucosa have dense, homogenous, aporous collagen structures that potentially limit cellular penetration, undermining the theoretical benefit of a "natural" collagen scaffold. We hypothesized that Miromesh-a novel prosthetic derived from porcine liver by perfusion decellularization-provides a more optimal matrix for tissue ingrowth. METHODS: Thirty rats underwent survival surgery that constituted the creation of a 4 × 1 cm abdominal defect and simultaneous bridged repair. Twenty rats were bridged with Miromesh, and 10 rats were bridged with non-cross-linked porcine dermis (Strattice). Ten Miromesh and all 10 Strattice were rinsed in vancomycin solution and inoculated with 10(4) colony-forming units of green fluorescent protein-labeled Staphylococcus aureus (GFP-SA) after implantation. Ten Miromesh controls were neither soaked nor inoculated. No animals received systemic antibiotics. All animals were euthanized at 90 d and underwent an examination of their gross appearance before being sectioned for quantitative bacterial culture and histologic grading. A pathologist scored specimens (0-4) for cellular infiltration, acute inflammation, chronic inflammation, granulation tissue, foreign body reaction, and fibrous capsule formation. RESULTS: All but one rat repaired with Strattice survived until the 90-d euthanization. All quantitative bacterial cultures for inoculated specimens were negative for GFP-SA. Of nine Strattice explants, none received a cellular infiltration score >0, consistent with a poor tissue-mesh interface observed grossly. Of 10 Miromesh explants also inoculated with GFP-SA, seven of 10 demonstrated cellular infiltration with an average score of +2.7 ± 0.8, whereas sterile Miromesh implants received an average score of 0.8 ± 1.0. Two inoculated Miromesh implants demonstrated acute inflammation and infection on histology. CONCLUSIONS: A prosthetic generated from porcine liver by perfusion decellularization provides a matrix for superior cellular infiltration compared with non-cross-linked porcine dermis.

Cell-coating affects tissue integration of synthetic and biologic meshes: comparative analysis of the onlay and underlay mesh positioning in rats.

BACKGROUND: While mesh reinforcement is recognized as the optimal strategy for many hernia repairs, there remains debate on the optimal position for deployment and characteristics that lead to improved biocompatibility. Coatings are an avenue by which integration may be improved. Our aim was to evaluate tissue integration between uncoated, fibroblast- and mesenchymal stem cell-coated meshes placed as subcutaneous onlay (ON) or intraperitoneal underlay (UN). METHODS: Three commonly used biologic and synthetic hernia meshes were tested including Parietex, TIGR and Strattice. Each mesh was coated with rat kidney fibroblasts (NRKs) or rat mesenchymal stem cells (MSCs) along with an uncoated group. In the ON group, mesh was fixated on top of the external oblique fascia. In the UN group, mesh was placed against the intact peritoneum. Animals were survived for 30 days and killed for biomechanical and histologic analysis. A "T"-peel test was performed on a mesh-tissue explant from each sample to analyze the strength of integration at the mesh-tissue interface. Tissue integration was evaluated histologically using an established scoring system. RESULTS: All uncoated meshes demonstrated significantly higher tissue ingrowth in the UN compared to ON position. Cell-coating of synthetic meshes decreased tissue ingrowth as UN, but increased it as ON, with a net effect of minimizing biomechanical difference between the two positions. In the biologic group however, NRK-coating decreased tissue ingrowth regardless of position, while MSC-coating increased it in both ON and UN positions. CONCLUSIONS: Both cell-coating and positioning affect mesh-tissue integration. Integration is superior in the underlay position compared to onlay when uncoated. Cell-coating of selected synthetic meshes can improve integration, particularly in the onlay position. Furthermore, MSCs appear to be a viable choice for biologic mesh coating, especially when implanted as an onlay. Overall, cell-coating of surgical meshes appears to a have a potential to improve mesh-tissue integration.

Epithelial barrier assembly requires coordinated activity of multiple domains of the tight junction protein ZO-1.

Tight junctions (TJs) regulate the paracellular movement of ions, macromolecules and immune cells across epithelia. Zonula occludens (ZO)-1 is a multi-domain polypeptide required for the assembly of TJs. MDCK II cells lacking ZO-1, and its homolog ZO-2, have three distinct phenotypes: reduced localization of occludin and some claudins to the TJs, increased epithelial permeability, and expansion of the apical actomyosin contractile array found at the apical junction complex (AJC). However, it is unclear exactly which ZO-1 binding domains are required to coordinate these activities. We addressed this question by examining the ability of ZO-1 domain-deletion transgenes to reverse the effects of ZO depletion. We found that the SH3 domain and the U5 motif are required to recruit ZO-1 to the AJC and that localization is a prerequisite for normal TJ and cytoskeletal organization. The PDZ2 domain is not required for localization of ZO-1 to the AJC, but is necessary to establish the characteristic continuous circumferential band of ZO-1, occludin and claudin-2. PDZ2 is also required to establish normal permeability, but is not required for normal cytoskeletal organization. Finally, our results demonstrate that PDZ1 is crucial for the normal organization of both the TJ and the AJC cytoskeleton. Our results establish that ZO-1 acts as a true scaffolding protein and that the coordinated activity of multiple domains is required for normal TJ structure and function.

Exploiting the inflammatory response on biomaterials research and development.

Exploiting the inflammatory response on biomaterials research and development requires an in-depth understanding of the cellular and molecular mechanisms involved in the events comprising the tissue response continuum. Examples of how the biomaterial surface chemistry may modulate the foreign body reaction to biomaterials. The utilization of different surface chemistries on biomaterials may provide biological design criteria for the appropriate use and function of biomaterials when used in medical devices and prostheses.