Deubiquitinating enzyme VCIP135 dictates the duration of botulinum neurotoxin type A intoxication.

Botulism is characterized by flaccid paralysis, which can be caused by intoxication with any of the seven known serotypes of botulinum neurotoxin (BoNT), all of which disrupt synaptic transmission by endoproteolytic cleavage of SNARE proteins. BoNT serotype A (BoNT/A) has the most prolonged or persistent effects, which can last several months, and exerts its effects by specifically cleaving and inactivating SNAP25. A major factor contributing to the persistence of intoxication is the long half-life of the catalytic light chain, which remains enzymatically active months after entry into cells. Here we report that BoNT/A catalytic light chain binds to, and is a substrate for, the ubiquitin ligase HECTD2. However, the light chain evades proteasomal degradation by the dominant effect of a deubiquitinating enzyme, VCIP135/VCPIP1. This deubiquitinating enzyme binds BoNT/A light chain directly, with the two associating in cells through the C-terminal 77 amino acids of the light chain protease. The development of specific DUB inhibitors, together with inhibitors of BoNT/A proteolytic activity, may be useful for reducing the morbidity and public health costs associated with BoNT/A intoxication and could have potential biodefense implications.

Algal chloroplast produced camelid VH H antitoxins are capable of neutralizing botulinum neurotoxin.

We have produced three antitoxins consisting of the variable domains of camelid heavy chain-only antibodies (VH H) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen-binding proteins and the heterodimer fusion protein containing two VH H domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin-producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.

Chlorovirus Skp1-binding ankyrin repeat protein interplay and mimicry of cellular ubiquitin ligase machinery.

UNLABELLED: The ubiquitin-proteasome system is targeted by many viruses that have evolved strategies to redirect host ubiquitination machinery. Members of the genus Chlorovirus are proposed to share an ancestral lineage with a broader group of related viruses, nucleo-cytoplasmic large DNA viruses (NCLDV). Chloroviruses encode an Skp1 homolog and ankyrin repeat (ANK) proteins. Several chlorovirus-encoded ANK repeats contain C-terminal domains characteristic of cellular F-boxes or related NCLDV chordopox PRANC (pox protein repeats of ankyrin at C-terminal) domains. These observations suggested that this unique combination of Skp1 and ANK repeat proteins might form complexes analogous to the cellular Skp1-Cul1-F-box (SCF) ubiquitin ligase complex. We identified two ANK proteins from the prototypic chlorovirus Paramecium bursaria chlorella virus-1 (PBCV-1) that functioned as binding partners for the virus-encoded Skp1, proteins A682L and A607R. These ANK proteins had a C-terminal Skp1 interactional motif that functioned similarly to cellular F-box domains. A C-terminal motif of ANK protein A682L binds Skp1 proteins from widely divergent species. Yeast two-hybrid analyses using serial domain deletion constructs confirmed the C-terminal localization of the Skp1 interactional motif in PBCV-1 A682L. ANK protein A607R represents an ANK family with one member present in all 41 sequenced chloroviruses. A comprehensive phylogenetic analysis of these related ANK and viral Skp1 proteins suggested partnered function tailored to the host alga or common ancestral heritage. Here, we show protein-protein interaction between corresponding family clusters of virus-encoded ANK and Skp1 proteins from three chlorovirus types. Collectively, our results indicate that chloroviruses have evolved complementing Skp1 and ANK proteins that mimic cellular SCF-associated proteins. IMPORTANCE: Viruses have evolved ways to direct ubiquitination events in order to create environments conducive to their replication. As reported in the manuscript, the large chloroviruses encode several components involved in the SCF ubiquitin ligase complex including a viral Skp1 homolog. Studies on how chloroviruses manipulate their host algal ubiquitination system will provide insights toward viral protein mimicry, substrate recognition, and key interactive domains controlling selective protein degradation. These findings may also further understanding of the evolution of other large DNA viruses, like poxviruses, that are reported to share the same monophyly lineage as chloroviruses.

Bioprospecting of microalgae for integrated biomass production and phytoremediation of unsterilized wastewater and anaerobic digestion centrate.

Eighteen microalgae, including two local isolates, were evaluated for their ability to grow and remove nutrients from unsterilized primary or secondary wastewater effluents as well as wastewater supplemented with nutrient-rich anaerobic digester centrate (ADC). Most of the tested species except several phylogenetically clustered Chlorella sorokiniana including local isolates and Scenedesmus strains were unable to grow efficiently. This may reflect the presence of certain genetic traits important for robust growth in the unsterilized wastewater. The maximum algal-specific growth rates and biomass density obtained in these bacterial-contaminated cultures were in the range of 0.8-1 day(-1) and 250-350 mg L(-1), respectively. ADC supplementation was especially helpful to biologically treated secondary effluent with its lower initial macronutrient and micronutrient content. As a result of algal growth, total nitrogen and orthophosphate levels were reduced by as much as 90 and 70 %, respectively. Biological assimilation was estimated to be the main mechanism of nitrogen removal in primary and secondary effluents with ammonia volatilization and bacterial nitrification-denitrification contributing for cultures supplemented with ADC. Assimilation by algae served as the principal mechanism of orthophosphate remediation in secondary wastewater cultures, while chemical precipitation appeared also to be important for orthophosphate removal in primary wastewater. Overall, cultivation of microalgae in primary and primary + 5 % ADC may be more favorable from an economical and sustainability perspective due to elimination of the costly and energy-intensive biological treatment step. These findings demonstrate that unsterilized wastewater and ADC can serve as critical nutrient sources for biomass generation and that robust microalgae can be potent players in wastewater phytoremediation.

Generation of a phage-display library of single-domain camelid VH H antibodies directed against Chlamydomonas reinhardtii antigens, and characterization of VH Hs binding cell-surface antigens.

Single-domain antibodies (sdAbs) are powerful tools for the detection, quantification, purification and subcellular localization of proteins of interest in biological research. We have generated camelid (Lama pacos) heavy chain-only variable VH domain (VH H) libraries against antigens in total cell lysates from Chlamydomonas reinhardtii. The sdAbs in the sera from immunized animals and VH H antibody domains isolated from the library show specificity to C. reinhardtii and lack of reactivity to antigens from four other algae: Chlorella variabilis, Coccomyxa subellipsoidea, Nannochloropsis oceanica and Thalassiosira pseudonana. Antibodies were produced against a diverse representation of antigens as evidenced by sera ELISA and protein-blot analyses. A phage-display library consisting of the VH H region contained at least 10(6) individual transformants, and thus should represent a wide range of C. reinhardtii antigens. The utility of the phage library was demonstrated by using live C. reinhardtii cells to pan for VH H clones with specific recognition of cell-surface epitopes. The lead candidate VH H clones (designated B11 and H10) bound to C. reinhardtii with EC50 values ≤ 0.5 nm. Treatment of cells with VH H B11 fused to the mCherry or green fluorescent proteins allowed brilliant and specific staining of the C. reinhardtii cell wall and analysis of cell-wall genesis during cell division. Such high-complexity VH H antibody libraries for algae will be valuable tools for algal researchers and biotechnologists.

Expanding the spectral palette of fluorescent proteins for the green microalga Chlamydomonas reinhardtii.

Fluorescent proteins (FPs) have become essential tools for a growing number of fields in biology. However, such tools have not been widely adopted for use in microalgal research. The aim of this study was to express and compare six FPs (blue mTagBFP, cyan mCerulean, green CrGFP, yellow Venus, orange tdTomato and red mCherry) in the popular model microalga Chlamydomonas reinhardtii. To circumvent the transgene silencing that often occurs in C. reinhardtii, the FPs were expressed from the nuclear genome as transcriptional fusions with the sh-ble antibiotic resistance gene, with the foot and mouth disease virus 2A self-cleaving sequence placed between the coding sequences. All ble-2A-FPs tested are well-expressed and efficiently processed to yield mature, unfused FPs that localize throughout the cytoplasm. The fluorescence signals of each FP were detectable in whole cells by fluorescence microplate reader analysis, live-cell fluorescence microscopy, and flow cytometry. Furthermore, we report a comparative analysis of fluorescence levels relative to auto-fluorescence for the chosen FPs. Finally, we demonstrate that the ble-2A expression vector may be used to fluorescently label an endogenous protein (α-tubulin). We show that the mCerulean-α-tubulin fusion protein localizes to the cytoskeleton and flagella, as expected, and that cells containing this fusion protein had normal cellular function. Overall, our results indicate that, by use of the ble-2A nuclear expression construct, a wide array of FP tools and technologies may be applied to microalgal research, opening up many possibilities for microalgal biology and biotechnology.

A rapid live-cell ELISA for characterizing antibodies against cell surface antigens of Chlamydomonas reinhardtii and its use in isolating algae from natural environments with related cell wall components.

BACKGROUND: Cell walls are essential for most bacteria, archaea, fungi, algae and land plants to provide shape, structural integrity and protection from numerous biotic and abiotic environmental factors. In the case of eukaryotic algae, relatively little is known of the composition, structure or mechanisms of assembly of cell walls in individual species or between species and how these differences enable algae to inhabit a great diversity of environments. In this paper we describe the use of camelid antibody fragments (VHHs) and a streamlined ELISA assay as powerful new tools for obtaining mono-specific reagents for detecting individual algal cell wall components and for isolating algae that share a particular cell surface component. RESULTS: To develop new microalgal bioprospecting tools to aid in the search of environmental samples for algae that share similar cell wall and cell surface components, we have produced single-chain camelid antibodies raised against cell surface components of the single-cell alga, Chlamydomonas reinhardtii. We have cloned the variable-region domains (VHHs) from the camelid heavy-chain-only antibodies and overproduced tagged versions of these monoclonal-like antibodies in E. coli. Using these VHHs, we have developed an accurate, facile, low cost ELISA that uses live cells as a source of antigens in their native conformation and that requires less than 90 minutes to perform. This ELISA technique was demonstrated to be as accurate as standard ELISAs that employ proteins from cell lysates and that generally require >24 hours to complete. Among the cloned VHHs, VHH B11, exhibited the highest affinity (EC50 < 1 nM) for the C. reinhardtii cell surface. The live-cell ELISA procedure was employed to detect algae sharing cell surface components with C. reinhardtii in water samples from natural environments. In addition, mCherry-tagged VHH B11 was used along with fluorescence activated cell sorting (FACS) to select individual axenic isolates of presumed wild relatives of C. reinhardtii and other Chlorphyceae from the same environmental samples. CONCLUSIONS: Camelid antibody VHH domains provide a highly specific tool for detection of individual cell wall components of algae and for allowing the selection of algae that share a particular cell surface molecule from diverse ecosystems.

Persistence of Botulinum neurotoxin inactivation of nerve function.

The extraordinary persistence of intoxication occurring after exposure to some Botulinum neurotoxin (BoNT) serotypes is both a therapeutic marvel and a biodefense nightmare. Understanding the mechanisms underlying BoNT persistence will offer new strategies for improving the efficacy and extending the applications of BoNT therapeutic agents as well as for treating the symptoms of botulism. Research indicates that the persistence of BoNT intoxication can be influenced both by the ability of the toxin protease or its cleaved soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein substrate to resist turnover. Protease turnover seems to be mediated in part by the ubiquitin-proteasome system (UPS) and efforts to manipulate the UPS may prove to be an effective strategy for improving therapeutic utility of BoNT products and in the development of botulism antidotes.

The effect of iron on growth, lipid accumulation, and gene expression profile of the freshwater microalga Chlorella sorokiniana.

The effects of iron on the growth, lipid accumulation, and gene expression profiles of the limnetic Chlorella sorokiniana CCTCC M209220 under photoautotrophy were investigated. The addition of iron up to 10(-5) mol l(-l) increased final cell densities by nearly 2-fold at 2.3 × 10(7) cells/ml, growth rate by 2-fold, and the length of the exponential phase by 5 days as compared to unsupplemented controls while 10(-3) mol l(-1) iron was toxic. The lipid content increased from 12 % for unsupplemented cultures to 33 % at 10(-4) mol l(-1) iron while the highest overall lipid yield reached 179 mg l(-1). A genefishing and qPCR comparison between the C. sorokiniana at low and high iron levels indicated increases in the expression of several genes, including carbonic anhydrase involved in microalgal cell growth, as well as acc1 and choline transporter related to lipid synthesis. This study provides insights into changes in gene expression and metabolism that accompany iron supplementation to Chlorella as well as potential metabolic engineering targets for improving growth and lipid synthesis in microalgae.

Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system.

Botulinum neurotoxins (BoNTs) are the most potent natural toxins known. The effects of BoNT serotype A (BoNT/A) can last several months, whereas the effects of BoNT serotype E (BoNT/E), which shares the same synaptic target, synaptosomal-associated protein 25 (SNAP25), last only several weeks. The long-lasting effects or persistence of BoNT/A, although desirable for therapeutic applications, presents a challenge for medical treatment of BoNT intoxication. Although the mechanisms for BoNT toxicity are well known, little is known about the mechanisms that govern the persistence of the toxins. We show that the recombinant catalytic light chain (LC) of BoNT/E is ubiquitylated and rapidly degraded in cells. In contrast, BoNT/A LC is considerably more stable. Differential susceptibility of the catalytic LCs to ubiquitin-dependent proteolysis therefore might explain the differential persistence of BoNT serotypes. In this regard we show that TRAF2, a RING finger protein implicated in ubiquitylation, selectively associates with BoNT/E LC and promotes its proteasomal degradation. Given these data, we asked whether BoNT/A LC could be targeted for rapid proteasomal degradation by redirecting it to characterized ubiquitin ligase domains. We describe chimeric SNAP25-based ubiquitin ligases that target BoNT/A LC for degradation, reducing its duration in a cellular model for toxin persistence.

Physiological evaluation of a new Chlorella sorokiniana isolate for its biomass production and lipid accumulation in photoautotrophic and heterotrophic cultures.

A novel green unicellular microalgal isolate from the freshwater of the Inner Mongolia Province of China and named as CCTCC M209220, grows between pH 6 and 11 and temperatures of 20-35°C with optimal conditions at pH 9 and 30°C. Morphological features and the phylogenetic analysis for the 18S rRNA gene reveal that the isolate is a Chlorella sorokiniana strain. A nitrogen source test reveals that this strain can grow well with nitrate and urea, but not ammonium. The strain can grow heterotrophically with glucose as the carbon source and accumulates lipid content as high as 56% (w/w) dry weight after 7 days in high glucose concentrations compared to 19% lipids achieved in 30 days of photoautotrophic culture. The relative neutral lipid content as a fraction of the total lipid is also much higher in heterotrophic culture as compared to photoautotrophic culture.

The effect of mixotrophy on microalgal growth, lipid content, and expression levels of three pathway genes in Chlorella sorokiniana.

Nannochloropsis oculata CCMP 525, Dunaliella salina FACHB 435, and Chlorella sorokiniana CCTCC M209220 were compared in mixotrophic and photoautotrophic cultures in terms of growth rate, protein, and lipid content. Growth improved in glucose, and the biomass productivities of N. oculata, D. salina, and C. sorokiniana were found to be 1.4-, 2.2- and 4.2-fold that observed photoautotrophically. However, biomass and lipid production decreased at the highest glucose concentrations. Meanwhile, the content of protein and lipid were significantly augmented for mixotrophic conditions at least for some species. C. sorokiniana was found to be well suited for lipid production based on its high biomass production rate and lipid content reaching 51% during mixotrophy. Expression levels of accD (heteromeric acetyl-CoA carboxylase beta subunit), acc1 (homomeric acetyl-CoA carboxylase), rbcL (ribulose 1, 5-bisphosphate carboxylase/oxygenase large subunit) genes in C. sorokiniana were studied by real-time PCR. Increased expression levels of accD reflect the increased lipid content in stationary phase of mixotrophic growth, but expression of the acc1 gene remains low, suggesting that this gene may not be critical to lipid accumulation. Additionally, reduction of expression of the rbcL gene during mixotrophy indicated that utilization of glucose was found to reduce the role of this gene and photosynthesis.

Protracted renal clearance of fentanyl in persons with opioid use disorder.

INTRODUCTION: The illicit opioid supply in the U.S. is increasingly adulterated with fentanyl. As such, persons with opioid use disorder (OUD) may be regularly exposed to fentanyl, however, the pharmacokinetics of repeated fentanyl exposure are not well understood. The current study aimed to quantify renal clearance of fentanyl in OUD patients presenting to residential treatment. METHODS: Participants (N = 12) who presented to a 28-day residential treatment program were enrolled if they tested positive for fentanyl at intake. Urine samples were collected every 2-3 days and were quantitatively tested for fentanyl, norfentanyl, and creatinine via liquid chromatography mass spectrometry (LC-MS). Fentanyl clearance was defined as the time since last illicit opioid use and the median time between last positive and first negative fentanyl urine screen. RESULTS: Participants had a mean and standard deviation (SD) age of 28.9 (11.0), were 67 % male, and 83 % white. The mean (SD) time for fentanyl and norfentanyl clearance was 7.3 (4.9) and 13.3 (6.9) days, respectively. One participant continued to test positive for fentanyl for 19 days and norfentanyl for 26 days following their last use, and left treatment without testing negative for norfentanyl. CONCLUSION: Fentanyl clearance in persons with OUD is considerably longer than the typical 2-4 day clearance of other short-acting opioids. The findings of this study might explain recent reports of difficulty in buprenorphine inductions for persons who use fentanyl, and point to a need to better understand the pharmacokinetics of fentanyl in the context of opioid withdrawal in persons who regularly use fentanyl.

Differences in Availability and Use of Medications for Opioid Use Disorder in Residential Treatment Settings in the United States.

Importance: While many individuals with opioid use disorder seek treatment at residential facilities to initiate long-term recovery, the availability and use of medications for opioid use disorder (MOUDs) in these facilities is unclear. Objective: To examine differences in MOUD availability and use in residential facilities as a function of Medicaid policy, facility-level factors associated with MOUD availability, and admissions-level factors associated with MOUD use. Design, Setting, and Participants: This cross-sectional study used deidentified facility-level and admissions-level data from 2863 residential treatment facilities and 232 414 admissions in the United States in 2017. Facility-level data were extracted from the 2017 National Survey of Substance Abuse Treatment Services, and admissions-level data were extracted from the 2017 Treatment Episode Data Set-Admissions. Statistical analyses were conducted from June to November 2019. Exposures: Admissions for opioid use disorder at residential treatment facilities in the United States that identified opioids as the patient's primary drug of choice. Main Outcomes and Measures: Availability and use of 3 MOUDs (ie, extended-release naltrexone, buprenorphine, and methadone). Results: Of 232 414 admissions, 205 612 (88.5%) contained complete demographic data (166 213 [80.8%] aged 25-54 years; 136 854 [66.6%] men; 151 867 [73.9%] white). Among all admissions, MOUDs were used in only 34 058 of 192 336 (17.7%) in states that expanded Medicaid and 775 of 40 078 (1.9%) in states that did not expand Medicaid (P < .001). A relatively low percentage of the 2863 residential treatment facilities in this study offered extended-release naltrexone (854 [29.8%]), buprenorphine (953 [33.3%]), or methadone (60 [2.1%]). Compared with residential facilities that offered at least 1 MOUD, those that offered no MOUDs had lower odds of also offering psychiatric medications (odds ratio [OR], 0.06; 95% CI, 0.05-0.08; Wald χ21 = 542.09; P < .001), being licensed by a state or hospital authority (OR, 0.39; 95% CI, 0.27-0.57; Wald χ21 = 24.28; P < .001), or being accredited by a health organization (OR, 0.28; 95% CI, 0.23-0.33; Wald χ21 = 180.91; P < .001). Residential facilities that did not offer any MOUDs had higher odds of accepting cash-only payments than those that offered at least 1 MOUD (OR, 4.80; 95% CI, 3.47-6.64; Wald χ21 = 89.65; P < .001). Conclusions and Relevance: In this cross-sectional study of residential addiction treatment facilities in the United States, MOUD availability and use were sparse. Public health and policy efforts to improve access to and use of MOUDs in residential treatment facilities could improve treatment outcomes for individuals with opioid use disorder who are initiating recovery.

An improved in vitro and in vivo Sindbis virus expression system through host and virus engineering.

The Sindbis viral expression system enables the rapid production of high levels of recombinant protein in mammalian cells; however, this expression is typically limited to transient production due to the cytotoxicity of the virus. Limiting the lethality inherent in the Sindbis virus vector in order to enable long term, sustained expression of recombinant proteins may be possible. In this study, modifications to virus and host have been combined in order to reduce the cytopathic effects. Non-cytopathic replication competent viruses of two Sindbis viral strains, TE and 633, were developed using a non-structural protein (nsP) P726S point mutation in order to obtain persistent heterologous gene expression in infected Baby Hamster Kidney (BHK) cells and Chinese Hamster Ovary (CHO) cells. Cells infected with the P726S variant viruses were able to recover after infection, while cells infected with normal virus died within 3 days. The P726S mutation did not reduce the susceptibility of 5- and 14-day-old mice to 633 and TE viruses in vivo. In addition, animal survival with the P726S variant viruses was increased and GFP expression was sustained for at least 14 days while the 633 and TE infection resulted in short-term GFP expression or an earlier mortality. Modifications to the host BHK and CHO cells themselves were subsequently undertaken by including the anti-apoptotic gene Bcl-2 and a deletion mutant of Bcl-2 (Bcl-2Delta) as another method for limiting the cytopathic effects of the Sindbis virus. The inclusion of anti-apoptotic genes permitted higher production of heterologous GFP protein following Sindbis virus infection, and the combination of the TE-P726S virus and the CHO-Bcl-2Delta cell line showed the greatest improvement in cell survival. Sindbis virus infection also induced ER stress in mammalian cells as detected by increased PERK phosphorylation and ATF4 translation. Overexpression of Parkin, an E3 ubiquitin ligase that can protect cells against agents that induce ER stress, suppressed Sindbis virus-induced cell death in both BHK cells and in vivo studies in mice. Such findings show that viral and host modifications can improve cell survival and production of heterologous proteins, change viral behavior in vitro and in vivo, and assist in the development of new expression or gene delivery vehicles.

A green light for engineered algae: redirecting metabolism to fuel a biotechnology revolution.

Microalgae have the potential to revolutionize biotechnology in a number of areas including nutrition, aquaculture, pharmaceuticals, and biofuels. Although algae have been commercially cultivated for over 50 years, metabolic engineering now seems necessary in order to achieve their full processing capabilities. Recently, the development of a number of transgenic algal strains boasting recombinant protein expression, engineered photosynthesis, and enhanced metabolism encourage the prospects of designer microalgae. Given the vast contributions that these solar-powered, carbon dioxide-sequestering organisms can provide to current global markets and the environment, an intensified focus on microalgal biotechnology is warranted. Ongoing advances in cultivation techniques coupled with genetic manipulation of crucial metabolic networks will further promote microalgae as an attractive platform for the production of numerous high-value compounds.

Trends in first-time treatment admissions for older adults with alcohol use disorder: Availability of medical and specialty clinical services in hospital, residential, and outpatient facilities.

BACKGROUND: Alcohol use disorder (AUD) is a growing problem among older adults. The aim of this study was to quantify trends in first-time treatment admissions for older adults with AUD in the U.S., and examine the medical and specialty clinical services offered by treatment facility type. METHODS: Patient level data were collected from the Treatment Episode Data Set for Admissions between 2004-2017. Joinpoint regression was used to identify unique trends in first-time treatment admissions for older adults with AUD. Provider level data were collected from the National Survey of Substance Abuse Treatment Services (N-SSATS) for the most recent year, 2017. N-SSATS data were grouped by facility type (inpatient/hospital, residential, and outpatient treatment) to examine differences in medications and clinical services. RESULTS: Among all persons seeking first-time treatment for AUD with alcohol as their primary drug of choice (n = 3,606,948), there was a significant increase in the proportion of older adults seeking treatment from 2004 to 2017 (p-trend<0.001), with an average annual percent change of 6.8% (95% confidence intervals: 6.2%-7.4%). The majority of older adults with AUD sought treatment in outpatient and residential facilities, which compared to hospital-based facilities had lower odds of offering supervised detoxification, acamprosate, naltrexone, psychiatric medications, or mental health services (all p-values<0.001). Fewer than 25% of hospital-based and 20% of residential or outpatient facilities offered specialty services for older adults. CONCLUSIONS: U.S. substance abuse treatment providers are not compensating for the changing nature of admissions by older adults, and are not providing state of the art services for this population.

The C-terminal transmembrane domain of Bcl-xL mediates changes in mitochondrial morphology.

We investigate the effect of mitochondrial localization and the Bcl-x(L) C-terminal transmembrane (TM) domain on mitochondrial morphology and subcellular light scattering. CSM 14.1 cell lines stably expressed yellow fluorescent protein (YFP), YFP-Bcl-x(L,) YFP-Bcl-x(L)-DeltaTM, containing the remainder of Bcl-x(L) after deletion of the last 21 amino acids corresponding to the TM domain, or YFP-TM, consisting of YFP fused at its C-terminal to the last 21 amino acids of Bcl-x(L). YFP-Bcl-x(L) and YFP-TM localized to the mitochondria. Their expression decreased the intensity ratio of wide-to-narrow angle forward scatter by subcellular organelles, and correlated with an increase in the proportion of mitochondria with an expanded matrix having greatly reduced intracristal spaces as observed by electron microscopy. Cells expressing YFP-TM also exhibited significant autophagy. In contrast, YFP-Bcl-x(L)-DeltaTM was diffusely distributed in the cells, and its expression did not alter light scattering or mitochondrial morphology compared with parental cells. Expression of YFP-Bcl-x(L) or YFP-Bcl-x(L)-DeltaTM provided significant resistance to staurosporine-induced apoptosis. Surprisingly however, YFP-TM expression also conferred a moderate level of cell death resistance in response to staurosporine. Taken together, our results suggest the existence of a secondary Bcl-x(L) function that is mediated by the transmembrane domain, alters mitochondrial morphology, and is distinct from BH3 domain sequestration.

E2F-1 overexpression increases viable cell density in batch cultures of Chinese hamster ovary cells.

The cell density is an inherent constraint in commercial mammalian cell cultures. Here, we describe a cell engineering strategy utilizing the overexpression of the E2F-1 cell cycle transcription factor in CHO DG44 cells that produce a monoclonal antibody in serum-free, suspension culture. Stable pools and cell lines expressing E2F-1 were isolated that attained viable cell densities 20% higher than control cell lines and continued proliferation for an additional day in batch culture. There were no significant changes in antibody production, apoptosis, and cell cycle compared to control cells, nor were the growth effects evident in fed-batch conditions. Overall, E2F-1 overexpression postponed entry into stationary phase in mammalian cells, but perhaps novel E2F-1 variants or combination cell cycle engineering strategies will be necessary to realize significant growth benefits in commercial applications.

Evolution of DDB1-binding WD40 (DWD) in the viridiplantae.

Damaged DNA Binding 1 (DDB1)-binding WD40 (DWD) proteins are highly conserved and involved in a plethora of developmental and physiological processes such as flowering time control, photomorphogenesis, and abiotic stress responses. The phylogeny of this family of proteins in plants and algae of viridiplante is a critical area to understand the emergence of this family in such important and diverse functions. We aimed to investigate the putative homologs of DWD in the viridiplante and establish a deeper DWD evolutionary grasp. The advancement in publicly available genomic data allowed us to perform an extensive genome-wide DWD retrieval. Using annotated Arabidopsis thaliana DWDs as the reference, we generated and characterized a comprehensive DWD database for the studied photoautotrophs. Further, a generic DWD classification system (Type A to K), based on (i) position of DWD motifs, (ii) number of DWD motifs, and (iii) presence/absence of other domains, was adopted. About 72-80% DWDs have one DWD motif, whereas 17-24% DWDs have two and 0.5-4.7% DWDs have three DWD motifs. Neighbor-joining phylogenetic construction of A. thaliana DWDs facilitated us to tune these substrate receptors into 15 groups. Though the DWD count increases from microalgae to higher land plants, the ratio of DWD to WD40 remained constant throughout the viridiplante. The DWD expansion appeared to be the consequence of consistent DWD genetic flow accompanied by several gene duplication events. The network, phylogenetic, and statistical analysis delineated DWD evolutionary relevance in the viridiplante.