Effect of osteophyte removal on simulated range of motion using 3-dimensional preoperative planning software for reverse total shoulder arthroplasty.

Background: Glenohumeral osteophytes (OPs) can adversely influence postoperative range of motion (ROM) following shoulder arthroplasty due to mechanical impingement. Though commercial three-dimensional preoperative planning software (3D PPS) is available to simulate ROM before and after OP resection, little is known about the magnitude of effect OPs and their subsequent removal have on simulated glenohumeral ROM. Methods: Included patients were 1) indicated for reverse total shoulder arthroplasty (rTSA) using 3D PPS and 2) presented with glenoid and/or humeral head OPs on preoperative two-dimensional computed tomography (2D-CT) imaging. Thirty patients met the inclusion criteria (9 females, 21 males; mean age 70.45 ± 4.99 years, range 63-80 years). All subjects (n = 30) presented with humeral OPs (mean volume: 2905.16 mm3, range 109.1-11,246 mm3), while 11 subjects also presented with glenoid OPs (mean volume 108.06 mm3, range 37.59-791.4 mm3). Preoperative CTs were used to calculate OP volume (mm3) and OP circumferential extent (clockface). Mean clockface position for circumferential humeral OPs originated at 6:09 (range 4:30-7:15) and extended to 8:51 (range 8:15-10:15). Mean clockface position for glenoid OPs originated at 3:00 (range 2:00-5:00) and extended to 6:16 (range 3:00-7:30). 3D implants on PPS were standardized to achieve 0° of version, 0° of inclination and 4 mm of net lateralization. Thirty-nine and thirty-six mm glenospheres were used for males and females, respectively. 3D PPS was used to evaluate simulated ROM differences before and after OP removal in the planes of adduction (ADD), abduction, internal rotation (IR), external rotation (ER), extension, and flexion. Impact of OP volume and circumferential extent on pre and postop removal ROM were also analyzed. Results: Humeral OP removal significantly increased impingement-free ADD, IR, ER, extension, and flexion. Removal of larger (mm3) humeral OPs positively correlated with improvement in IR (R = 0.452, P = .011), ER (R = 0.394, P = .033), and flexion (R = 0.500, P < .01). Greater circumferential extent of humeral OPs correlated with worse preremoval ROM in the planes of ADD (R = 0.364, P = .02) and extension (R = 0.403, P = .04), and improvements in ER postop removal (R = 0.431, P = .03). Conclusion: Humeral OP removal significantly increases impingement-free ADD, IR, ER, extension, and flexion in simulated 3D PPS models following rTSA. Magnitude of simulated ROM improvement is influenced by initial humeral OP volume and circumferential clockface extent. Surgeons should consider these effects when using 3D PPS for rTSA planning to optimize postoperative ROM prognostics.

Epidemiologic Changes in Pediatric Fractures Presenting to Emergency Departments During the COVID-19 Pandemic.

BACKGROUND: Fractures are a common pediatric injury. The coronavirus disease 2019 (COVID-19) pandemic resulted in significant changes in daily life that could impact the incidence of pediatric fractures. The purpose of this study was to compare the incidence of pediatric fractures in the United States during the COVID-19 pandemic to previous seasonally adjusted fracture incidence rates using the National Electronic Injury Surveillance System (NEISS) database and the American Community Survey (ACS). METHODS: The NEISS database was queried from 2016 to 2020 for fractures occurring in pediatric (0 to 17 y) patients. ACS population data allowed for the estimation of fracture incidence per 1000 person-years. Using a quasiexperimental interrupted time series design, Poisson regression models were constructed to test the overall and differential impact of COVID-19 on monthly fracture rate by age, sex, fracture site, injury location, and disposition. RESULTS: Our sample consisted of 121,803 cases (mean age 9.6±4.6 y, 36.1% female) representing 2,959,421±372,337 fractures nationally. We identified a stable 27% decrease in fractures per month after February 2020 [risk difference (RD) per 1000 youth years=-2.3; 95% confidence interval: -2.98, -1.57]). We found significant effect modification by age, fracture site and injury location ( P <0.05). The fracture incidence among children 5 years or older significantly decreased, as well as the incidence of fractures at school [RD=-0.96 (-1.09, -0.84)] and during sports [risk difference=-1.55 (-1.77, -1.32)]. There was also a trend toward a reduction in upper extremity fractures and fractures requiring admission. CONCLUSION: A nationally representative injury database demonstrated a 27% decline in monthly pediatric fractures during the COVID-19 pandemic that persisted into the latter half of 2020. These trends appeared most attributable to a reduction in fractures discharged home and upper extremity fractures among older children sustained at school and in sports. Our findings provide unique insight into the epidemiology of pediatric fractures and demonstrate a baseline need for musculoskeletal care among young children even in the setting of a national shutdown. LEVEL OF EVIDENCE: Level II-retrospective prognostic study.

Differential expression patterns of phospholipase D isoforms 1 and 2 in the mammalian brain and retina.

Phosphatidic acid is a key signaling molecule heavily implicated in exocytosis due to its protein-binding partners and propensity to induce negative membrane curvature. One phosphatidic acid-producing enzyme, phospholipase D (PLD), has also been implicated in neurotransmission. Unfortunately, due to the unreliability of reagents, there has been confusion in the literature regarding the expression of PLD isoforms in the mammalian brain which has hampered our understanding of their functional roles in neurons. To address this, we generated epitope-tagged PLD1 and PLD2 knockin mice using CRISPR/Cas9. Using these mice, we show that PLD1 and PLD2 are both localized at synapses by adulthood, with PLD2 expression being considerably higher in glial cells and PLD1 expression predominating in neurons. Interestingly, we observed that only PLD1 is expressed in the mouse retina, where it is found in the synaptic plexiform layers. These data provide critical information regarding the localization and potential role of PLDs in the central nervous system.

Subscapularis Repair Prior to Subscapularis Takedown in Anatomic Shoulder Arthroplasty: Improving Anatomic Restoration and Mechanics of the Subscapularis.

Traditionally, total shoulder arthroplasty is performed using a deltopectoral approach through which the glenohumeral joint is accessed by mobilization of the subscapularis. Despite several variations on the subscapularis management techniques, postoperative complications, including subscapularis deficiency and lower functional outcomes, remain an area for improvement. The purpose of this Technical Note is to describe in detail our technique for management of the subscapularis in the setting of a stemless humeral implant through which the repair is planned and almost entirely performed at the beginning of the case, prior to the subscapularis peel. This technique aims to improve outcomes after total shoulder arthroplasty by 1) avoiding the anatomic implant with anchor drilling, 2) improving procedure efficiency, and 3) anatomically "repairing" the subscapularis prior to takedown by placing anchors exactly at the repair-tension site.

What is the deviation in 3D preoperative planning software? A systematic review of concordance between plan and actual implant in reverse total shoulder arthroplasty.

BACKGROUND: Three-dimensional (3D) preoperative planning software for reverse total shoulder arthroplasty (rTSA) has been implemented in recent years in order to increase accuracy, improve efficiency, and add value to the outcome. A comprehensive literature review is required to determine the utility of preoperative 3D planning software in guiding orthopedic surgeons for implant placement in rTSA. We hypothesize that implementation of 3D preoperative planning software in the setting of rTSA leads to high concordance with minimal deviation from the preoperative plan. METHODS: A comprehensive and iterative literature review was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) using the PubMed, Embase, OVID Medline, Scopus, Cinahl, Web of Science, and Cochrane databases for original English-language studies evaluating the impact of preoperative planning software usage on rTSA outcomes published from January 1, 2000, to present. Blinded reviewers conducted multiple screens. All included studies were graded based on level of evidence, and data concerning patient demographics and postoperative outcomes were extracted. RESULTS: Nine articles met inclusion criteria (1 level II, 3 level III, and 5 level IV articles), including 415 patients and 422 shoulders. Of the patients who underwent rTSA, 235 were female and 140 were male, although 3 studies (n = 40) did not report sex breakdowns for rTSA patients. The average age was 72.7 years. Four studies (79 shoulders) reported implant final position as mean deviation from planned version and planned inclination. Six studies (n = 236) reported screw angle deviation, fixation, length, and concordance. Concordance with the preoperative plan was measured in 3 studies (n = 178), resulting in complete concordance of 90% (n = 100), arthroplasty type concordance (rTSA vs. TSA) of 100% (n = 100), and glenosphere size concordance between 93% (n = 100) and 88% (n = 76). For screw length concordance, baseplate screw matched by 81% (n = 76) and 100% (n = 2), and upper (n = 35) and lower (n = 35) screw length concordance was observed as 74% and 69%, respectively. The use of preoperative planning (n = 178) was associated with low deviation from preoperative plan, more 2-screw fixations, and longer average screw length in comparison with an unplanned cohort. CONCLUSION: The use of preoperative planning software in the setting of rTSA results in minimal deviation from preoperative plan. High levels of concordance in screw angle, screw length, and glenosphere size were observed. Further prospective studies should be conducted to further substantiate these results.

Impact of fatty infiltration of the rotator cuff on reverse total shoulder arthroplasty outcomes: a systematic review.

Background and hypothesis: The impact of preoperative fatty infiltration of specific rotator cuff muscles on the outcomes of reverse total shoulder arthroplasty (rTSA) has not been well defined. Preoperative fatty infiltration of the shoulder musculature will negatively affect rTSA outcomes. Methods: A comprehensive literature review was conducted as per the Preferred Reporting Items for Systematic Reviews and Meta-analyses using PubMed, Embase, OVID Medline, Scopus, Cinahl, Web of Science, and Cochrane databases for original, English-language studies evaluating effect of fatty infiltration of shoulder musculature on rTSA outcomes published from January 1, 2000 to present. Blinded reviewers conducted multiple screens. All included studies were graded based on the level of evidence, and data concerning patient demographics and postoperative outcomes were extracted. Results: A total of 11 articles were included, including one level I article, three level III articles, and seven level IV articles. The review consisted of 720 patients and 731 shoulders (320 women and 157 men), with a mean age of 72.4 years. A single deltopectoral approach was performed for a majority of studies (627/731 shoulders), followed by a superolateral approach (70/731 shoulders) and a single transdeltoid approach (4/731 patients). Eleven studies reported data specifically about preoperative fatty infiltration of the rotator cuff musculature; the teres minor was studied most widely (298/731 shoulders), followed by the subscapularis (256/731 shoulders) and infraspinatus (232/731 shoulders). The Constant score (562/731 shoulders) and American Shoulder and Elbow Surgeons score (284/731 shoulders) were the most common recorded outcome scores. Fatty infiltration of the teres minor, supraspinatus, and infraspinatus was associated with worse range of motion after rTSA. Conclusion: Preoperative fatty infiltration of the rotator cuff, particularly of the teres minor and infraspinatus, has a negative impact on subjective patient outcomes and restoration of range of motion, especially external rotation, after rTSA. The impact of fatty infiltration of the other rotator cuff muscles remains unclear, which may be due to intersurgeon differences in the handling of the remaining rotator cuff muscles or differences in implant design. The evaluated literature provides information on which patients can be educated about probable outcomes and restoration of function after rTSA.

Alveolar, Endothelial, and Organ Injury Marker Dynamics in Severe COVID-19.

Rationale: Alveolar and endothelial injury may be differentially associated with coronavirus disease (COVID-19) severity over time. Objectives: To describe alveolar and endothelial injury dynamics and associations with COVID-19 severity, cardiorenovascular injury, and outcomes. Methods: This single-center observational study enrolled patients with COVID-19 requiring respiratory support at emergency department presentation. More than 40 markers of alveolar (including receptor for advanced glycation endproducts [RAGE]), endothelial (including angiopoietin-2), and cardiorenovascular injury (including renin, kidney injury molecule-1, and troponin-I) were serially compared between invasively and spontaneously ventilated patients using mixed-effects repeated-measures models. Ventilatory ratios were calculated for intubated patients. Associations of biomarkers with modified World Health Organization scale at Day 28 were determined with multivariable proportional-odds regression. Measurements and Main Results: Of 225 patients, 74 (33%) received invasive ventilation at Day 0. RAGE was 1.80-fold higher in invasive ventilation patients at Day 0 (95% confidence interval [CI], 1.50-2.17) versus spontaneous ventilation, but decreased over time in all patients. Changes in alveolar markers did not correlate with changes in endothelial, cardiac, or renal injury markers. In contrast, endothelial markers were similar to lower at Day 0 for invasive ventilation versus spontaneous ventilation, but then increased over time only among intubated patients. In intubated patients, angiopoietin-2 was similar (fold difference, 1.02; 95% CI, 0.89-1.17) to nonintubated patients at Day 0 but 1.80-fold higher (95% CI, 1.56-2.06) at Day 3; cardiorenovascular injury markers showed similar patterns. Endothelial markers were not consistently associated with ventilatory ratios. Endothelial markers were more often significantly associated with 28-day outcomes than alveolar markers. Conclusions: Alveolar injury markers increase early. Endothelial injury markers increase later and are associated with cardiorenovascular injury and 28-day outcome. Alveolar and endothelial injury likely contribute at different times to disease progression in severe COVID-19.

Revision Anterior Cruciate Ligament, Lateral Collateral Ligament Reconstruction, and Osteochondral Allograft Transplantation for Complex Knee Instability.

Anterior cruciate ligament (ACL) injuries rarely occur as an isolated event and often include associated meniscal, subchondral bone, and collateral ligament injuries. Concomitant pathology frequently complicates primary and revision ACL reconstruction and must be addressed to ensure comprehensive diagnosis and treatment. In this Technical Note, we describe our method for treatment of complex knee instability following multiple failed ACL reconstruction using a multiligament reconstruction technique with an osteochondral allograft transplantation to the lateral femoral condyle. This comprehensive repair technique restores the anatomic load bearing forces of the cruciate and collateral ligaments and promotes biological repair through incorporation of cartilage resurfacing to ultimately achieve optimal kinematics of the knee joint.

Early cross-coronavirus reactive signatures of humoral immunity against COVID-19.

The introduction of vaccines has inspired hope in the battle against SARS-CoV-2. However, the emergence of viral variants, in the absence of potent antivirals, has left the world struggling with the uncertain nature of this disease. Antibodies currently represent the strongest correlate of immunity against SARS-CoV-2, thus we profiled the earliest humoral signatures in a large cohort of acutely ill (survivors and nonsurvivors) and mild or asymptomatic individuals with COVID-19. Although a SARS-CoV-2­specific immune response evolved rapidly in survivors of COVID-19, nonsurvivors exhibited blunted and delayed humoral immune evolution, particularly with respect to S2-specific antibodies. Given the conservation of S2 across ß-coronaviruses, we found that the early development of SARS-CoV-2­specific immunity occurred in tandem with preexisting common ß-coronavirus OC43 humoral immunity in survivors, which was also selectively expanded in individuals that develop a paucisymptomatic infection. These data point to the importance of cross-coronavirus immunity as a correlate of protection against COVID-19.

SARS-CoV-2 viremia is associated with distinct proteomic pathways and predicts COVID-19 outcomes.

BACKGROUNDSARS-CoV-2 plasma viremia has been associated with severe disease and death in COVID-19 in small-scale cohort studies. The mechanisms behind this association remain elusive.METHODSWe evaluated the relationship between SARS-CoV-2 viremia, disease outcome, and inflammatory and proteomic profiles in a cohort of COVID-19 emergency department participants. SARS-CoV-2 viral load was measured using a quantitative reverse transcription PCR-based platform. Proteomic data were generated with Proximity Extension Assay using the Olink platform.RESULTSThis study included 300 participants with nucleic acid test-confirmed COVID-19. Plasma SARS-CoV-2 viremia levels at the time of presentation predicted adverse disease outcomes, with an adjusted OR of 10.6 (95% CI 4.4-25.5, P < 0.001) for severe disease (mechanical ventilation and/or 28-day mortality) and 3.9 (95% CI 1.5-10.1, P = 0.006) for 28-day mortality. Proteomic analyses revealed prominent proteomic pathways associated with SARS-CoV-2 viremia, including upregulation of SARS-CoV-2 entry factors (ACE2, CTSL, FURIN), heightened markers of tissue damage to the lungs, gastrointestinal tract, and endothelium/vasculature, and alterations in coagulation pathways.CONCLUSIONThese results highlight the cascade of vascular and tissue damage associated with SARS-CoV-2 plasma viremia that underlies its ability to predict COVID-19 disease outcomes.FUNDINGMark and Lisa Schwartz; the National Institutes of Health (U19AI082630); the American Lung Association; the Executive Committee on Research at Massachusetts General Hospital; the Chan Zuckerberg Initiative; Arthur, Sandra, and Sarah Irving for the David P. Ryan, MD, Endowed Chair in Cancer Research; an EMBO Long-Term Fellowship (ALTF 486-2018); a Cancer Research Institute/Bristol Myers Squibb Fellowship (CRI2993); the Harvard Catalyst/Harvard Clinical and Translational Science Center (National Center for Advancing Translational Sciences, NIH awards UL1TR001102 and UL1TR002541-01); and by the Harvard University Center for AIDS Research (National Institute of Allergy and Infectious Diseases, 5P30AI060354).

Large-scale phenotypic drug screen identifies neuroprotectants in zebrafish and mouse models of retinitis pigmentosa.

Retinitis pigmentosa (RP) and associated inherited retinal diseases (IRDs) are caused by rod photoreceptor degeneration, necessitating therapeutics promoting rod photoreceptor survival. To address this, we tested compounds for neuroprotective effects in multiple zebrafish and mouse RP models, reasoning drugs effective across species and/or independent of disease mutation may translate better clinically. We first performed a large-scale phenotypic drug screen for compounds promoting rod cell survival in a larval zebrafish model of inducible RP. We tested 2934 compounds, mostly human-approved drugs, across six concentrations, resulting in 113 compounds being identified as hits. Secondary tests of 42 high-priority hits confirmed eleven lead candidates. Leads were then evaluated in a series of mouse RP models in an effort to identify compounds effective across species and RP models, that is, potential pan-disease therapeutics. Nine of 11 leads exhibited neuroprotective effects in mouse primary photoreceptor cultures, and three promoted photoreceptor survival in mouse rd1 retinal explants. Both shared and complementary mechanisms of action were implicated across leads. Shared target tests implicated parp1-dependent cell death in our zebrafish RP model. Complementation tests revealed enhanced and additive/synergistic neuroprotective effects of paired drug combinations in mouse photoreceptor cultures and zebrafish, respectively. These results highlight the value of cross-species/multi-model phenotypic drug discovery and suggest combinatorial drug therapies may provide enhanced therapeutic benefits for RP patients.

Photoreceptors are the cells responsible for vision. They are part of the retina: the light-sensing tissue at the back of the eye. They come in two types: rods and cones. Rods specialise in night vision, while cones specialise in daytime colour vision. The death of these cells can cause a disease, called retinitis pigmentosa, that leads to vision loss. Symptoms often start in childhood with a gradual loss of night vision. Later on, loss of cone photoreceptors can lead to total blindness. Unfortunately, there are no treatments available that protect photoreceptor cells from dying. Research has identified drugs that can protect photoreceptors in animal models, but these drugs have failed in humans. The classic way to look for new treatments is to find drugs that target molecules implicated in a disease, and then test them to see if they are effective. Unfortunately, many drugs identified in this way fail in later stages of testing, either because they are ineffective, or because they have unacceptable side effects. One way to reverse this trend is to first test whether a drug is effective at curing a disease in animals, and later determining what it does at a molecular level. This could reveal whether drugs can protect photoreceptors before research to discover their molecular targets begins. Tests like this across different species could maximise the chances of finding a drug that works in humans, because if a drug works in several species, it is more likely to have shared target molecules across species. Applying this reasoning, Zhang et al. tested around 3,000 drug candidates for treating retinitis pigmentosa in a strain of zebrafish that undergoes photoreceptor degeneration similar to the human disease. Most of these drug candidates already have approval for use in humans, meaning that if they were found to be effective for treating retinitis pigmentosa, they could be fast-tracked for use in people. Zhang et al. found three compounds that helped photoreceptors survive both in zebrafish and in retinas grown in the laboratory derived from a mouse strain with degeneration similar to retinitis pigmentosa. Tests to find out how these three compounds worked at the molecular level revealed that they interfered with a protein that can trigger cell death. The tests also found other promising compounds, many of which offered increased protection when combined in pairs. Worldwide there are between 1.5 and 2.5 million people with retinitis pigmentosa. With this disease, loss of vision happens slowly, so identifying drugs that could slow or stop the process could help many people. These results suggest that placing animal testing earlier in the drug discovery process could complement traditional target-based methods. The compounds identified here, and the information about how they work, could expand potential treatment research. The next step in this research is to test whether the drugs identified by Zhang et al. protect mammals other than mice from the degeneration seen in retinitis pigmentosa.

Longitudinal proteomic analysis of severe COVID-19 reveals survival-associated signatures, tissue-specific cell death, and cell-cell interactions.

Mechanisms underlying severe coronavirus disease 2019 (COVID-19) disease remain poorly understood. We analyze several thousand plasma proteins longitudinally in 306 COVID-19 patients and 78 symptomatic controls, uncovering immune and non-immune proteins linked to COVID-19. Deconvolution of our plasma proteome data using published scRNA-seq datasets reveals contributions from circulating immune and tissue cells. Sixteen percent of patients display reduced inflammation yet comparably poor outcomes. Comparison of patients who died to severely ill survivors identifies dynamic immune-cell-derived and tissue-associated proteins associated with survival, including exocrine pancreatic proteases. Using derived tissue-specific and cell-type-specific intracellular death signatures, cellular angiotensin-converting enzyme 2 (ACE2) expression, and our data, we infer whether organ damage resulted from direct or indirect effects of infection. We propose a model in which interactions among myeloid, epithelial, and T cells drive tissue damage. These datasets provide important insights and a rich resource for analysis of mechanisms of severe COVID-19 disease.

Genetic access to neurons in the accessory optic system reveals a role for Sema6A in midbrain circuitry mediating motion perception.

The accessory optic system (AOS) detects retinal image slip and reports it to the oculomotor system for reflexive image stabilization. Here, we characterize two Cre lines that permit genetic access to AOS circuits responding to vertical motion. The first (Pcdh9-Cre) labels only one of the four subtypes of ON direction-selective retinal ganglion cells (ON-DS RGCs), those preferring ventral retinal motion. Their axons diverge from the optic tract just behind the chiasm and selectively innervate the medial terminal nucleus (MTN) of the AOS. Unlike most RGC subtypes examined, they survive after optic nerve crush. The second Cre-driver line (Pdzk1ip1-Cre) labels postsynaptic neurons in the MTN. These project predominantly to the other major terminal nucleus of the AOS, the nucleus of the optic tract (NOT). We find that the transmembrane protein semaphorin 6A (Sema6A) is required for the formation of axonal projections from the MTN to the NOT, just as it is for the retinal innervation of the MTN. These new tools permit manipulation of specific circuits in the AOS and show that Sema6A is required for establishing AOS connections in multiple locations.

LKB1 and AMPK regulate synaptic remodeling in old age.

Age-related decreases in neural function result in part from alterations in synapses. To identify molecular defects that lead to such changes, we focused on the outer retina, in which synapses are markedly altered in old rodents and humans. We found that the serine/threonine kinase LKB1 and one of its substrates, AMPK, regulate this process. In old mice, synaptic remodeling was accompanied by specific decreases in the levels of total LKB1 and active (phosphorylated) AMPK. In the absence of either kinase, young adult mice developed retinal defects similar to those that occurred in old wild-type animals. LKB1 and AMPK function in rod photoreceptors where their loss leads to aberrant axonal retraction, the extension of postsynaptic dendrites and the formation of ectopic synapses. Conversely, increasing AMPK activity genetically or pharmacologically attenuates and may reverse age-related synaptic alterations. Together, these results identify molecular determinants of age-related synaptic remodeling and suggest strategies for attenuating these changes.

SAD kinases control the maturation of nerve terminals in the mammalian peripheral and central nervous systems.

Axons develop in a series of steps, beginning with specification, outgrowth, and arborization, and terminating with formation and maturation of presynaptic specializations. We found previously that the SAD-A and SAD-B kinases are required for axon specification and arborization in subsets of mouse neurons. Here, we show that following these steps, SAD kinases become localized to synaptic sites and are required within presynaptic cells for structural and functional maturation of synapses in both peripheral and central nervous systems. Deleting SADs from sensory neurons can perturb either axonal arborization or nerve terminal maturation, depending on the stage of deletion. Thus, a single pair of kinases plays multiple, sequential roles in axonal differentiation.

Lkb1/Stk11 regulation of mTOR signaling controls the transition of chondrocyte fates and suppresses skeletal tumor formation.

Liver kinase b1 (Lkb1) protein kinase activity regulates cell growth and cell polarity. Here, we show Lkb1 is essential for maintaining a balance between mitotic and postmitotic cell fates in development of the mammalian skeleton. In this process, Lkb1 activity controls the progression of mitotic chondrocytes to a mature, postmitotic hypertrophic fate. Loss of this Lkb1-dependent switch leads to a dramatic expansion of immature chondrocytes and formation of enchondroma-like tumors. Pathway analysis points to a mammalian target of rapamycin complex 1-dependent mechanism that can be partially suppressed by rapamycin treatment. These findings highlight a critical requirement for integration of mammalian target of rapamycin activity into developmental decision-making during mammalian skeletogenesis.

SAD-A kinase controls islet ß-cell size and function as a mediator of mTORC1 signaling.

The mammalian target of rapamycin (mTOR) plays an important role in controlling islet ß-cell function. However, the underlying molecular mechanisms remain poorly elucidated. Synapses of amphids defective kinase-A (SAD-A) is a 5' adenosine monophosphate-activated protein kinase-related protein kinase that is exclusively expressed in pancreas and brain. In this study, we investigated a role of the kinase in regulating pancreatic ß-cell morphology and function as a mediator of mTOR complex 1 (mTORC1) signaling. We show that global SAD-A deletion leads to defective glucose-stimulated insulin secretion and petite islets, which are reminiscent of the defects in mice with global deletion of ribosomal protein S6 kinase 1, a downstream target of mTORC1. Consistent with these findings, selective deletion of SAD-A in pancreas decreased islet ß-cell size, whereas SAD-A overexpression significantly increased the size of mouse insulinomas cell lines ß-cells. In direct support of SAD-A as a unique mediator of mTORC1 signaling in islet ß-cells, we demonstrate that glucose dramatically stimulated SAD-A protein translation in isolated mouse islets, which was potently inhibited by rapamycin, an inhibitor of mTORC1. Moreover, the 5'-untranslated region of SAD-A mRNA is highly structured and requires mTORC1 signaling for its translation initiation. Together, these findings identified SAD-A as a unique pancreas-specific effector protein of mTORC1 signaling.

SAD kinases sculpt axonal arbors of sensory neurons through long- and short-term responses to neurotrophin signals.

Extrinsic cues activate intrinsic signaling mechanisms to pattern neuronal shape and connectivity. We showed previously that three cytoplasmic Ser/Thr kinases, LKB1, SAD-A, and SAD-B, control early axon-dendrite polarization in forebrain neurons. Here, we assess their role in other neuronal types. We found that all three kinases are dispensable for axon formation outside of the cortex but that SAD kinases are required for formation of central axonal arbors by subsets of sensory neurons. The requirement for SAD kinases is most prominent in NT-3 dependent neurons. SAD kinases transduce NT-3 signals in two ways through distinct pathways. First, sustained NT-3/TrkC signaling increases SAD protein levels. Second, short-duration NT-3/TrkC signals transiently activate SADs by inducing dephosphorylation of C-terminal domains, thereby allowing activating phosphorylation of the kinase domain. We propose that SAD kinases integrate long- and short-duration signals from extrinsic cues to sculpt axon arbors within the CNS.

SAD-A potentiates glucose-stimulated insulin secretion as a mediator of glucagon-like peptide 1 response in pancreatic ß cells.

Type 2 diabetes is characterized by defective glucose-stimulated insulin secretion (GSIS) from pancreatic ß cells, which can be restored by glucagon-like peptide 1 (GLP-1), an incretin hormone commonly used for the treatment of type 2 diabetes. However, molecular mechanisms by which GLP-1 affects glucose responsiveness in islet ß cells remain poorly understood. Here we investigated a role of SAD-A, an AMP-activated protein kinase (AMPK)-related kinase, in regulating GSIS in mice with conditional SAD-A deletion. We show that selective deletion of SAD-A in pancreas impaired incretin's effect on GSIS, leading to glucose intolerance. Conversely, overexpression of SAD-A significantly enhanced GSIS and further potentiated GLP-1's effect on GSIS from isolated mouse islets. In support of SAD-A as a mediator of incretin response, SAD-A is expressed exclusively in pancreas and brain, the primary targeting tissues of GLP-1 action. Additionally, SAD-A kinase is activated in response to stimulation by GLP-1 through cyclic AMP (cAMP)/Ca(2+)-dependent signaling pathways in islet ß cells. Furthermore, we identified Thr443 as a key autoinhibitory phosphorylation site which mediates SAD-A's effect on incretin response in islet ß cells. Consequently, ablation of Thr443 significantly enhanced GLP-1's effect on GSIS from isolated mouse islets. Together, these findings identified SAD-A kinase as a pancreas-specific mediator of incretin response in islet ß cells.

Derlin-2-deficient mice reveal an essential role for protein dislocation in chondrocytes.

Protein quality control is a balance between chaperone-assisted folding and removal of misfolded proteins from the endoplasmic reticulum (ER). Cell-based assays have been used to identify key players of the dislocation machinery, including members of the Derlin family. We generated conditional knockout mice to examine the in vivo role of Derlin-2, a component that nucleates cellular dislocation machinery. In most Derlin-2-deficient tissues, we found constitutive upregulation of ER chaperones and IRE-1-mediated induction of the unfolded protein response. The IRE-1/XBP-1 pathway is required for development of highly secretory cells, particularly plasma cells and hepatocytes. However, B lymphocyte development and antibody secretion were normal in the absence of Derlin-2. Likewise, hepatocyte function was unaffected by liver-specific deletion of Derlin-2. Whole-body deletion of Derlin-2 results in perinatal death. The few mice that survived to adulthood all developed skeletal dysplasia, likely caused by defects in collagen matrix protein secretion by costal chondrocytes.