A requirement for cytoplasmic protein synthesis during chloroplast senescence in the aquatic plant Anacharis canadensis

Cycloheximide (CHI) at 1 µg/liter delayed the loss ofchlorophyll from detached Anacharis canadensis leaflets senescingin the dark. Chloramphenicol (CAP) and streptomycin (SM) slightlyaccelerated the loss. CHI was effective even during the laterstages of senescence in preventing further loss of chlorophyll.Senescence proceeded normally upon return of the leaflets intowater. The need for cytoplasmic protein synthesis during chloroplastsenescence and the types of proteins involved are discussed. (Received March 26, 1976; )     

SARS-CoV-2-specific T cells associate with inflammation and reduced lung function in pulmonary post-acute sequalae of SARS-CoV-2

As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had between 6- and 105-fold higher frequencies of IFN-γ- and TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells in peripheral blood, and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV¹), while increased frequencies of IFN-γ-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.     

Demographic responses to climate‐driven variation in habitat quality across the annual cycle of a migratory bird species

The demography and dynamics of migratory bird populations depend on patterns of movement and habitat quality across the annual cycle. We leveraged archival GPS‐tagging data, climate data, remote‐sensed vegetation data, and bird‐banding data to better understand the dynamics of black‐headed grosbeak (Pheucticus melanocephalus) populations in two breeding regions, the coast and Central Valley of California (Coastal California) and the Sierra Nevada mountain range (Sierra Nevada), over 28 years (1992–2019). Drought conditions across the annual cycle and rainfall timing on the molting grounds influenced seasonal habitat characteristics, including vegetation greenness and phenology (maturity dates). We developed a novel integrated population model with population state informed by adult capture data, recruitment rates informed by age‐specific capture data and climate covariates, and survival rates informed by adult capture–mark–recapture data and climate covariates. Population size was relatively variable among years for Coastal California, where numbers of recruits and survivors were positively correlated, and years of population increase were largely driven by recruitment. In the Sierra Nevada, population size was more consistent and showed stronger evidence of population regulation (numbers of recruits and survivors negatively correlated). Neither region showed evidence of long‐term population trend. We found only weak support for most climate–demographic rate relationships. However, recruitment rates for the Coastal California region were higher when rainfall was relatively early on the molting grounds and when wintering grounds were relatively cool and wet. We suggest that our approach of integrating movement, climate, and demographic data within a novel modeling framework can provide a useful method for better understanding the dynamics of broadly distributed migratory species.     

Amphiphiles for supercritical CO2

A semi-fluorinated hybrid amphiphile, pentadecafluoro-5-dodecyl (F7H4) sulfate, has been shown to form reversed micelles in dense CO₂; the aggregates evolve to form water-in-CO₂ (w/c) microemulsion droplets on addition of water. Aggregation structures in these w/c phases have been characterised by small-angle neutron scattering (SANS), showing the presence of cylindrical droplets, which change into dispersed lamellar phases at even higher water loadings. Other systems are also introduced, being high internal phase emulsions (HIPEs) with brine, and liquid and supercritical CO₂, stabilized by certain commercially available nonylphenol ethoxylates (Dow Tergitol NP-, and Huntsman Surfonic N- amphiphiles). These dispersions have been characterised by SANS for the first time. Quantitative analyses of the HIPEs SANS profiles show that they behave similarly to hydrocarbon-water emulsion analogues, with regard to total interfacial areas and the effects of amphiphile concentration on the underlying structures. Finally, the advantages and disadvantages of both approaches for controlling the physico-chemical properties of liquid/supercritical CO₂ in potential applications are compared and contrasted. These results highlight the importance of using specially designed CO₂-philic amphiphiles for generating self-assembly structures in dense CO₂.     

Mechanisms of mucosal and parenteral tuberculosis vaccinations: adenoviral-based mucosal immunization preferentially elicits sustained accumulation of immune protective CD4 and CD8 T cells within the airway lumen

The mechanisms underlying better immune protection by mucosal vaccination have remained poorly understood. In our current study we have investigated the mechanisms by which respiratory virus-mediated mucosal vaccination provides remarkably better immune protection against pulmonary tuberculosis than parenteral vaccination. A recombinant adenovirus-based tuberculosis (TB) vaccine expressing Mycobacterium tuberculosis Ag85A (AdAg85A) was administered either intranasally (i.n.) or i.m. to mice, and Ag-specific CD4 and CD8 T cell responses, including frequency, IFN-gamma production, and CTL, were examined in the spleen, lung interstitium, and airway lumen. Although i.m. immunization with AdAg85A led to activation of T cells, particularly CD8 T cells, in the spleen and, to a lesser extent, in the lung interstitium, it failed to elicit any T cell response in the airway lumen. In contrast, although i.n. immunization failed to effectively activate T cells in the spleen, it uniquely elicited higher numbers of Ag-specific CD4 and CD8 T cells in the airway lumen that were capable of IFN-gamma production and cytolytic activities, as assessed by an intratracheal in vivo CTL assay. These airway luminal T cells of i.n. immunized mice or splenic T cells of i.m. immunized mice, upon transfer locally to the lungs of naive SCID mice, conferred immune protection against M. tuberculosis challenge. Our study has demonstrated that the airway luminal T cell population plays an important role in immune protection against pulmonary TB, thus providing mechanistic insights into the superior immune protection conferred by respiratory mucosal TB vaccination.     

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.     

The solid phase attachment of fungal hyphae in an ELISA to screen for antifungal antibodies

A method to immobilize fungal hyphae onto the wells of 96-well microplates for use in an in-direct ELISA to screen for antifungal antibodies in sera and cell culture supernatants is described. The hyphae from three genera (Penicillium, Eurotium andFusarium) were successfully attached by overnight drying onto wells precoated with poly-L-lysine and glutaraldehyde. Microscopy revealed that the hyphae remained attached to the wells throughout the ELISA and antiserum titrations showed that the attached hyphae were uniformly coated and remained reactive. Background absorbances were low and the plates could be stored at –20 °C without loss of reactivity.     

Assessing strategies for improved superfamily recognition

There are more than 200 completed genomes and over 1 million nonredundant sequences in public repositories. Although the structural data are more sparse (approximately 13,000 nonredundant structures solved to date), several powerful sequence-based methodologies now allow these structures to be mapped onto related regions in a significant proportion of genome sequences. We review a number of publicly available strategies for providing structural annotations for genome sequences, and we describe the protocol adopted to provide CATH structural annotations for completed genomes. In particular, we assess the performance of several sequence-based protocols employing Hidden Markov model (HMM) technologies for superfamily recognition, including a new approach (SAMOSA [sequence augmented models of structure alignments]) that exploits multiple structural alignments from the CATH domain structure database when building the models. Using a data set of remote homologs detected by structure comparison and manually validated in CATH, a single-seed HMM library was able to recognize 76% of the data set. Including the SAMOSA models in the HMM library showed little gain in homolog recognition, although a slight improvement in alignment quality was observed for very remote homologs. However, using an expanded 1D-HMM library, CATH-ISL increased the coverage to 86%. The single-seed HMM library has been used to annotate the protein sequences of 120 genomes from all three major kingdoms, allowing up to 70% of the genes or partial genes to be assigned to CATH superfamilies. It has also been used to recruit sequences from Swiss-Prot and TrEMBL into CATH domain superfamilies, expanding the CATH database eightfold.