A unique AtSar1D-AtRabD2a nexus modulates autophagosome biogenesis in Arabidopsis thaliana

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1d-positive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis.

Autophagy is a conserved catabolic process characterized by the de novo generation of a double-membrane structure called an autophagosome with a fundamental function in the bulk turnover of cytoplasmic components, including proteins, RNAs, and organelles. Genetic studies in yeast have elucidated the molecular machinery of autophagy, whereby 42 autophagy-related (ATG) genes have been identified (1–3). These ATG genes are highly conserved among eukaryotes but often have multiple isoforms in other higher organisms, in particular in sessile plants. Albeit increasing understanding on the molecular function of Atg proteins in acting hierarchically on the phagophore assembly site (PAS) to produce autophagosomes, the origin of the autophagosomal membrane remains unclear in higher eukaryotes. Furthermore, the dedication of other membranes and machineries in the autophagy pathway remains under investigation.Plant autophagy is known to play important roles in the sessile lifestyle of plants, participating in seed germination, seedling establishment, plant development, hormone responses, lipid metabolism, and reproductive development (4). Plant autophagy research is advancing with findings not only on the counterparts of the yeast/mammalian Atg proteins but also dealing with some plant-unique factors functioning in different steps of autophagosome biogenesis, thereby uncovering novel mechanisms that might or might not be conserved in nonplant species (5). More interestingly, higher plants possess multiple protein isoforms of ATG machinery, whose functional heterogeneity in the autophagy pathway has only recently been unveiled (6).The coat protein complex II (COPII) machinery consists of five cytosolic components: the small GTPase Sar1, the inner coat protein dimer Sec23-Sec24, and the outer coat proteins Sec13-Sec31. These proteins are essential for COPII-coated vesicle formation, which buds from specialized regions of the ER, namely ER exit sites (ERESs) (7). Under nutrient-rich conditions, COPII vesicles mediate anterograde ER to Golgi transport. However, increasing evidence from yeast and mammals suggests that the COPII machinery or even COPII vesicles themselves may contribute to autophagosome formation when cells are starved for nutrients (8–16). Gene duplication events have occurred substantially in sessile plants during evolution, and the importance of distinct paralogs in environmental stress adaptation during plant development has been implied (17). Arabidopsis encodes multiple COPII paralogs in its genome, including five Sar1s, seven Sec23s, three Sec24s, two Sec13s, and two Sec31s (17). Increasing numbers of studies have pinpointed the functional diversity and importance of distinct COPII paralogs in ER protein export (18–23). Nonetheless, the mechanism by which COPII vesicles are redirected to the autophagy pathway upon nutrient starvation, and their roles in autophagosome biogenesis, remains unclear. Furthermore, the participation of specific COPII paralogs in autophagy regulation remains unknown in plants.Here, we report on a role of a specific Sar1 homolog, AtSar1d, that modulates plant autophagosome biogenesis in concert with AtRabD2a. Large-scale proteomic analysis of the ATG interactome has revealed possible mechanistic connections between the ATG machinery and specific COPII components in plants. Cellular and biochemical analyses have shown that the dominant negative (DN) mutant of AtSar1d (AtSar1dDN) specifically perturbs YFP-ATG8 vacuolar degradation upon autophagic induction. Consistently, a transfer DNA (T-DNA) insertion mutant of AtSar1d exhibited starvation-related phenotypes. Notably, AtSar1d regulates autophagosome progression through specific recognition of ATG8e by a previously uncharacterized noncanonical motif. We further identify a plant-unique Rab1/Ypt1 homolog AtRabD2a that colocalizes with AtSar1d and ATG8 upon starvation by transient expression in Arabidopsis protoplasts. A DN mutant of AtRabD2a (AtRabD2aNI) perturbs autophagy flux, while AtRabD2a is indispensable for bridging the AtSar1d-positive COPII vesicles with the ATG1 complex, thus contributing to autophagosome biogenesis in plants. Our study therefore unequivocally demonstrates that the plant-specific COPII machinery regulates autophagosome biogenesis and sheds light on the evolutionary importance of gene duplication events in the plant autophagy pathway.     

Value of preoperative investigations in diagnosing prosthetic joint infection: retrospective cohort study and literature review

The diagnosis of a prosthetic joint infection is difficult, but crucial for appropriate treatment. Scintigraphy with specific markers for infection (labelled white cells or immunoglobulin-G) has been reported as a more reliable diagnostic tool than clinical assessment (fever, fistula), laboratory studies (polynuclear neutrophil count, erythrocyte rate sedimentation, and C-reactive protein), and preoperative aspiration. In the first part of this study, we retrospectively reviewed 230 patients admitted with a suspected prosthetic joint infection, and examined the validity of the different diagnostic tools for the group as a whole and for subgroups according to the Coventry classification. In the second part, we reviewed 35 articles about preoperative evaluation of infection in prosthetic joints and compared them to our findings. Our study indicates that C-reactive protein and joint aspiration are the most useful tools to diagnose prosthetic joint infection even in situations of chronic infection (Coventry type II).     

The Diabetic Postoperative Mortality and Morbidity (DIPOM) trial: rationale and design of a multicenter, randomized, placebo-controlled, clinical trial of metoprolol for patients with diabetes mellitus who are undergoing major noncardiac surgery

Background

Recent trials suggest that perioperative β-blockade reduces the risk of cardiac events in patients with a risk of myocardial ischemia who are undergoing noncardiac surgery. Patients with diabetes mellitus are at a high-risk for postoperative cardiac morbidity and mortality. They may, therefore, benefit from perioperative β-blockade.

Methods

The Diabetic Postoperative Mortality and Morbidity (DIPOM) trial is an investigator-initiated and -controlled, centrally randomized, double-blind, placebo-controlled, multicenter trial. We compared the effect of metoprolol with placebo on mortality and cardiovascular morbidity rates in patients with diabetes mellitus who were β-blocker naive, ≥40 years old, and undergoing noncardiac surgery. The study drug was given during hospitalization for a maximum of 7 days beginning the evening before surgery. The primary outcome measure is the composite of all-cause mortality, acute myocardial infarction, unstable angina, or congestive heart failure leading to hospitalization or discovered or aggravated during hospitalization. Follow-up involves re-examination of patients at 6 months and collection of mortality and morbidity data via linkage to public databases. The study was powered on the basis of an estimated 30% 1-year event rate in the placebo arm and a 33% relative risk reduction in the metoprolol arm. The median follow-up period was 18 months.

Results

Enrollment started in July 2000 and ended in June 2002. A total of 921 patients were randomized, and 54% of these patients had known cardiac disease, hypertension, or both.

Conclusion

The results of this study may have implications for reduction of perioperative and postoperative risk in patients with diabetes mellitus who are undergoing major noncardiac surgery.     

A humid corridor across the Sahara for the migration of early modern humans out of Africa 120,000 years ago

It is widely accepted that modern humans originated in sub-Saharan Africa approximately 150-200 thousand years ago (ka), but their route of dispersal across the currently hyperarid Sahara remains controversial. Given that the first modern humans north of the Sahara are found in the Levant approximately 120-90 ka, northward dispersal likely occurred during a humid episode in the Sahara within Marine Isotope Stage (MIS) 5e (130-117 ka). The obvious dispersal route, the Nile, may be ruled out by notable differences between archaeological finds in the Nile Valley and the Levant at the critical time. Further west, space-born radar images reveal networks of-now buried-fossil river channels that extend across the desert to the Mediterranean coast, which represent alternative dispersal corridors. These corridors would explain scattered findings at desert oases of Middle Stone Age Aterian lithic industries with bifacial and tanged points that can be linked with industries further to the east and as far north as the Mediterranean coast. Here we present geochemical data that demonstrate that water in these fossil systems derived from the south during wet episodes in general, and penetrated all of the way to the Mediterranean during MIS 5e in particular. This proves the existence of an uninterrupted freshwater corridor across a currently hyperarid region of the Sahara at a key time for early modern human migrations to the north and out of Africa.     

Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu

Currently, no approved monoclonal antibody (mAb) therapies exist for human multiple myeloma (MM). Here we characterized cell surface CS1 as a novel MM antigen and further investigated the potential therapeutic utility of HuLuc63, a humanized anti-CS1 mAb, for treating human MM. CS1 mRNA and protein was highly expressed in CD138-purified primary tumor cells from the majority of MM patients (more than 97%) with low levels of circulating CS1 detectable in MM patient sera, but not in healthy donors. CS1 was expressed at adhesion-promoting uropod membranes of polarized MM cells, and short interfering RNA (siRNA) targeted to CS1 inhibited MM cell adhesion to bone marrow stromal cells (BMSCs). HuLuc63 inhibited MM cell binding to BMSCs and induced antibody-dependent cellular cytotoxicity (ADCC) against MM cells in dose-dependent and CS1-specific manners. HuLuc63 triggered autologous ADCC against primary MM cells resistant to conventional or novel therapies, including bortezomib and HSP90 inhibitor; and pretreatment with conventional or novel anti-MM drugs markedly enhanced HuLuc63-induced MM cell lysis. Administration of HuLuc63 significantly induces tumor regression in multiple xenograft models of human MM. These results thus define the functional significance of CS1 in MM and provide the preclinical rationale for testing HuLuc63 in clinical trials, either alone or in combination.     

Proxy‐reported health‐related quality of life of patients with juvenile idiopathic arthritis: The pediatric rheumatology international trials organization multinational quality of life cohort study

Objective

To investigate the proxy‐reported health‐related quality of life (HRQOL) and its determinants in patients with juvenile idiopathic arthritis (JIA).

Methods

In this multinational, multicenter, cross‐sectional study, HRQOL of patients with JIA was assessed through the Child Health Questionnaire (CHQ) and was compared with that of healthy children of similar age from the same geographic area. Potential determinants of HRQOL included demographic data, physician's and parent's global assessments, measures of joint inflammation, Childhood Health Assessment Questionnaire (CHAQ), and erythrocyte sedimentation rate.

Results

A total of 6,639 participants (3,324 with JIA and 3,315 healthy) were enrolled from 32 countries. The mean ± SD physical and psychosocial summary scores of the CHQ were significantly lower in patients with JIA than in healthy children (physical: 44.5 ± 10.6 versus 54.6 ± 4.0, P < 0.0001; psychosocial: 47.6 ± 8.7 versus 51.9 ± 7.5, P < 0.0001), with the physical well‐being domain being most impaired. Patients with persistent oligoarthritis had better HRQOL compared with other subtypes, whereas HRQOL was similar across patients with systemic arthritis, polyarthritis, and extended oligoarthritis. A CHAQ score >1 and a pain intensity rating >3.4 cm on a 10‐cm visual analog scale were the strongest determinants of poorer HRQOL in the physical and psychosocial domains, respectively.

Conclusion

We found that patients with JIA have a significant impairment of their HRQOL compared with healthy peers, particularly in the physical domain. Physical well‐being was mostly affected by the level of functional impairment, whereas the intensity of pain had the greatest influence on psychosocial health.     

Niche adaptation and genome expansion in the chlorophyll d-producing cyanobacterium Acaryochloris marina

Acaryochloris marina is a unique cyanobacterium that is able to produce chlorophyll d as its primary photosynthetic pigment and thus efficiently use far-red light for photosynthesis. Acaryochloris species have been isolated from marine environments in association with other oxygenic phototrophs, which may have driven the niche-filling introduction of chlorophyll d. To investigate these unique adaptations, we have sequenced the complete genome of A. marina. The DNA content of A. marina is composed of 8.3 million base pairs, which is among the largest bacterial genomes sequenced thus far. This large array of genomic data is distributed into nine single-copy plasmids that code for >25% of the putative ORFs. Heavy duplication of genes related to DNA repair and recombination (primarily recA) and transposable elements could account for genetic mobility and genome expansion. We discuss points of interest for the biosynthesis of the unusual pigments chlorophyll d and α-carotene and genes responsible for previously studied phycobilin aggregates. Our analysis also reveals that A. marina carries a unique complement of genes for these phycobiliproteins in relation to those coding for antenna proteins related to those in Prochlorococcus species. The global replacement of major photosynthetic pigments appears to have incurred only minimal specializations in reaction center proteins to accommodate these alternate pigments. These features clearly show that the genus Acaryochloris is a fitting candidate for understanding genome expansion, gene acquisition, ecological adaptation, and photosystem modification in the cyanobacteria.     

Muscular Architecture and Manometric Image of Gastroesophageal Barrier in the Rat

The two components of the gastroesophagealbarrier, the sphincter and the crural sling, closelyoverlap in humans, whereas they are widely separated inthe rat. This investigation correlates the anatomical components of the barrier and their manometriccounterparts in this animal. Sphincteric and cruralsling pressures were measured in four quadrants in 23rats. Muscle thickness was measured at nine levels of the gastroesophageal junction in the samequadrants in 12 rats and the muscular architecture ofthe region was studied in 10 fresh specimens. Themanometric sphincteric component is stronger on theright side where the thickest muscle fibers anchor tothe anterior and posterior borders of a mucosal ridgethat almost surround the cardia. Conversely, the slingpressure is highest towards the left where the muscular bundles straddle the esophagus. Inconclusion, there is a close correspondence between themanometric image and the muscular architecture of thecomponents of the gastroesophageal barrier in the rat. The anatomical arrangement of U-shapedmuscular bundles oriented in opposite directions createsa particularly powerful antireflux mechanism.