Robust lipid scrambling by TMEM16 proteins requires an open groove and thin membrane.

Biological membranes are complex and dynamic structures with different populations of lipids on their inner and outer leaflets. The Ca2+-activated TMEM16 family of membrane proteins play an important role in collapsing this asymmetric lipid distribution by spontaneously, and bidirectionally, scrambling phospholipids between the two leaflets, which can initiate signaling and alter the physical properties of the membrane. While evidence shows that lipid scrambling occurs via an open hydrophilic pathway ("groove") that spans the membrane, it remains unclear if all family members facilitate lipid movement in this manner. Here we present a comprehensive computational study of lipid scrambling by all TMEM16 members with experimentally solved structures. We performed coarse-grained molecular dynamics (MD) simulations of 27 structures from five different family members solved under activating and non-activating conditions, and we captured over 700 scrambling events in aggregate. This enabled us to directly compare scrambling rates, mechanisms, and protein-lipid interactions for fungal and mammalian TMEM16s, in both open (Ca2+-bound) and closed (Ca2+-free) conformations with statistical rigor. We show that nearly all (>90%) scrambling occurs in the dilated groove and the degree of induced membrane thinning positively correlates with the scrambling rate. Surprisingly, we also observed 60 scrambling events that occurred outside the canonical groove, over 90% of which took place at the dimer-dimer interface in mammalian TMEM16s. This new site suggests an alternative mechanism for lipid scrambling in the absence of an open groove.

Autoinhibition of a clamp-loader ATPase revealed by deep mutagenesis and cryo-EM.

Clamp loaders are AAA+ ATPases that facilitate high-speed DNA replication. In eukaryotic and bacteriophage clamp loaders, ATP hydrolysis requires interactions between aspartate residues in one protomer, present in conserved 'DEAD-box' motifs, and arginine residues in adjacent protomers. We show that functional defects resulting from a DEAD-box mutation in the T4 bacteriophage clamp loader can be compensated by widely distributed single mutations in the ATPase domain. Using cryo-EM, we discovered an unsuspected inactive conformation of the clamp loader, in which DNA binding is blocked and the catalytic sites are disassembled. Mutations that restore function map to regions of conformational change upon activation, suggesting that these mutations may increase DNA affinity by altering the energetic balance between inactive and active states. Our results show that there are extensive opportunities for evolution to improve catalytic efficiency when an inactive intermediate is involved.

Streptococcal 5'-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion.

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5'-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a Km of 169 µm and a Vmax of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5'-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg(2+), Ca(2+), or Mn(2+). However, Zn(2+) inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5'-nucleotidase activity and immune evasion properties.

Development of a new bioprocess scheme using frozen seed train intermediates to initiate CHO cell culture manufacturing campaigns.

Agility to schedule and execute cell culture manufacturing campaigns quickly in a multi-product facility will play a key role in meeting the growing demand for therapeutic proteins. In an effort to shorten campaign timelines, maximize plant flexibility and resource utilization, we investigated the initiation of cell culture manufacturing campaigns using CHO cells cryopreserved in large volume bags in place of the seed train process flows that are conventionally used in cell culture manufacturing. This approach, termed FASTEC (Frozen Accelerated Seed Train for Execution of a Campaign), involves cultivating cells to high density in a perfusion bioreactor, and cryopreserving cells in multiple disposable bags. Each run for a manufacturing campaign would then come from a thaw of one or more of these cryopreserved bags. This article reviews the development and optimization of individual steps of the FASTEC bioprocess scheme: scaling up cells to greater than 70 × 10(6) cells/mL and freezing in bags with an optimized controlled rate freezing protocol and using a customized rack configuration. Flow cytometry analysis was also employed to understand the recovery of CHO cells following cryopreservation. Extensive development data were gathered to ensure that the quantity and quality of the drug manufactured using the FASTEC bioprocess scheme was acceptable compared to the conventional seed train process flow. The result of offering comparable manufacturing options offers flexibility to the cell culture manufacturing network.

State vocational services and employment in cancer survivors.

BACKGROUND: This study investigated the association of state vocational rehabilitation services in the USA and work outcomes of cancer survivors who were unemployed prior to receipt of services. METHODS: Administrative data obtained during fiscal year 2005 from the Rehabilitation Services Administration (RSA) database consisting of 1,201 closed cases with the diagnosis of cancer formed the sample of this study. All cancer survivors were unemployed at the time of application. Data on demographic characteristics, employment and vocational service variables were extracted and analyzed in relation to employment outcome data. Multivariate logistic regression was used to examine the relationship among services provided and work outcomes accounting for demographic characteristics of the participants. RESULTS: Cancer survivors represented 0.4% of the total population that received vocational services in the state-federal vocational rehabilitation program. Of the unemployed cancer survivors who received services, 903 (57%) achieved successful employment while 670 (43%) were not employed following receipt of services. Gender (women; OR = 0.77, 95% CI = 0.61-0.97), lower educational levels (OR = 0.52, 95% CI = 0.33-0.81), provision of cash or medical benefits (e.g., Social Security Disability Insurance benefits; OR = 0.64, 95% CI = 0.50-0.82) were all associated with a greater likelihood of being unemployed at the end of vocational services. Counseling (OR = 1.33, 95% CI = 1.02-1.73), miscellaneous training (OR = 1.61, 95% CI = 1.06-2.44), rehabilitation technology services (OR = 1.22, 95% CI = 0.72-2.08), job placement services (OR = 2.37, 95% CI = 1.72-3.27), job search assistance (OR = 1.43; 95% CI = 1.02-2.01) maintenance services (OR = 1.92, 95% CI = 1.29-2.86), and other services (OR = 1.43, 95% CI = 1.07-1.90) were found to be significantly associated with increased odds for employment. CONCLUSION: Vocational rehabilitation services were found to be associated with employment status. Future studies investigating the specific effects of certain vocational services for unemployed cancer survivors who qualify for these services are warranted. IMPLICATIONS FOR CANCER SURVIVORS: Cancer survivors who are seeking employment or experiencing problems maintaining employment who can qualify should be encouraged to pursue services from state vocational rehabilitation agencies. Medical providers should also become familiar with services offered by state vocational rehabilitation agencies and consider the use of these services..