A Heme-based Redox Sensor in the Methanogenic Archaeon Methanosarcina acetivorans

Based on a bioinformatics study, the protein MA4561 from the methanogenic archaeon Methanosarcina acetivorans was originally predicted to be a multidomain phytochrome-like photosensory kinase possibly binding open-chain tetrapyrroles. Although we were able to show that recombinantly produced and purified protein does not bind any known phytochrome chromophores, UV-visible spectroscopy revealed the presence of a heme tetrapyrrole cofactor. In contrast to many other known cytoplasmic heme-containing proteins, the heme was covalently attached via one vinyl side chain to cysteine 656 in the second GAF domain. This GAF domain by itself is sufficient for covalent attachment. Resonance Raman and magnetic circular dichroism data support a model of a six-coordinate heme species with additional features of a five-coordination structure. The heme cofactor is redox-active and able to coordinate various ligands like imidazole, dimethyl sulfide, and carbon monoxide depending on the redox state. Interestingly, the redox state of the heme cofactor has a substantial influence on autophosphorylation activity. Although reduced protein does not autophosphorylate, oxidized protein gives a strong autophosphorylation signal independent from bound external ligands. Based on its genomic localization, MA4561 is most likely a sensor kinase of a two-component system effecting regulation of the Mts system, a set of three homologous corrinoid/methyltransferase fusion protein isoforms involved in methyl sulfide metabolism. Consistent with this prediction, an M. acetivorans mutant devoid of MA4561 constitutively synthesized MtsF. On the basis of our results, we postulate a heme-based redox/dimethyl sulfide sensory function of MA4561 and propose to designate it MsmS (methyl sulfide methyltransferase-associated sensor).     

Can a Shorter Psychomotor Vigilance Task Be Usedas a Reasonable Substitute for the Ten‐Minute Psychomotor Vigilance Task?

The 10 min psychomotor vigilance task (PVT) is commonly used in laboratory studies to assess the impact of sleep loss, sustained wakefulness, and/or time of day on neurobehavioral performance. In field settings, though, it may be impractical for participants to perform a test of this length. The aim of this study was to identify a performance measure that is sensitive to the effects of fatigue but less burdensome than a 10 min test. Sixteen participants (11 female, 5 male; mean age=21.7 years) slept in the sleep laboratory overnight then remained awake for 28 h from 08:00 h. During every second hour, participants completed three PVTs of differing duration (10 min, 5 min, 90 sec). For the 5 min/10 min comparison, ANOVA indicated that response time was significantly affected by test length (F_1,14=26.9, p<.001) and hours of wakefulness (F_13,182=46.1, p<.001) but not by their interaction (F_13,182=1.7, ns). There was a strong correlation between response time on the 5 and 10 min PVTs (r=.88, p<.001). For the 90 sec/10 min comparison, ANOVA indicated that response time was significantly affected by test length (F_1,14=65.9, p<.001) and hours of wakefulness (F_13,182=29.7, p<.001) as well as by their interaction (F_13,182=6.0, p<.001). There was a strong correlation between response time on the 90 sec and 10 min PVTs (r=.77, p<.001). The effects of hours of wakefulness on neurobehavioral performance were similar for the 5 min and 10 min PVTs. In contrast, performance on the 90 sec PVT was less affected by hours of wakefulness than on the 10 min PVT. In addition, performance on the 10 min PVT was more highly correlated with the 5 min PVT than the 90 sec PVT. These data indicate that the 5 min PVT may provide a reasonable substitute for the 10 min PVT in circumstances where a test shorter than 10 min is required.     

Aliphatic 1H, 13C and 15N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP+ and folate

Dihydrofolate reductase from the deep-sea bacterium Moritella profunda (MpDHFR) has been ¹³C/¹⁵N isotopically labelled and purified. Here, we report the aliphatic ¹H, ¹³C and ¹⁵N resonance assignments of MpDHFR in complex with NADP⁺ and folate. The spectra of MpDHFR suggest considerably greater conformational heterogeneity than is seen in the closely related DHFR from Escherichia coli.     

Clinical Application of Sleeping Beauty and Artificial Antigen Presenting Cells to Genetically Modify T Cells from Peripheral and Umbilical Cord Blood

The potency of clinical-grade T cells can be improved by combining gene therapy with immunotherapy to engineer a biologic product with the potential for superior (i) recognition of tumor-associated antigens (TAAs), (ii) persistence after infusion, (iii) potential for migration to tumor sites, and (iv) ability to recycle effector functions within the tumor microenvironment. Most approaches to genetic manipulation of T cells engineered for human application have used retrovirus and lentivirus for the stable expression of CAR^1-3. This approach, although compliant with current good manufacturing practice (GMP), can be expensive as it relies on the manufacture and release of clinical-grade recombinant virus from a limited number of production facilities. The electro-transfer of nonviral plasmids is an appealing alternative to transduction since DNA species can be produced to clinical grade at approximately 1/10^th the cost of recombinant GMP-grade virus. To improve the efficiency of integration we adapted Sleeping Beauty (SB) transposon and transposase for human application^4-8. Our SB system uses two DNA plasmids that consist of a transposon coding for a gene of interest (e.g. 2^nd generation CD19-specific CAR transgene, designated CD19RCD28) and a transposase (e.g. SB11) which inserts the transgene into TA dinucleotide repeats^9-11. To generate clinically-sufficient numbers of genetically modified T cells we use K562-derived artificial antigen presenting cells (aAPC) (clone #4) modified to express a TAA (e.g. CD19) as well as the T cell costimulatory molecules CD86, CD137L, a membrane-bound version of interleukin (IL)-15 (peptide fused to modified IgG4 Fc region) and CD64 (Fc-γ receptor 1) for the loading of monoclonal antibodies (mAb)¹². In this report, we demonstrate the procedures that can be undertaken in compliance with cGMP to generate CD19-specific CAR⁺ T cells suitable for human application. This was achieved by the synchronous electro-transfer of two DNA plasmids, a SB transposon (CD19RCD28) and a SB transposase (SB11) followed by retrieval of stable integrants by the every-7-day additions (stimulation cycle) of γ-irradiated aAPC (clone #4) in the presence of soluble recombinant human IL-2 and IL-21¹³. Typically 4 cycles (28 days of continuous culture) are undertaken to generate clinically-appealing numbers of T cells that stably express the CAR. This methodology to manufacturing clinical-grade CD19-specific T cells can be applied to T cells derived from peripheral blood (PB) or umbilical cord blood (UCB). Furthermore, this approach can be harnessed to generate T cells to diverse tumor types by pairing the specificity of the introduced CAR with expression of the TAA, recognized by the CAR, on the aAPC.     

A conserved asparagine residue in transmembrane segment 1 (TM1) of serotonin transporter dictates chloride-coupled neurotransmitter transport

Na(+)- and Cl(-)-dependent uptake of neurotransmitters via transporters of the SLC6 family, including the human serotonin transporter (SLC6A4), is critical for efficient synaptic transmission. Although residues in the human serotonin transporter involved in direct Cl(-) coordination of human serotonin transport have been identified, the role of Cl(-) in the transport mechanism remains unclear. Through a combination of mutagenesis, chemical modification, substrate and charge flux measurements, and molecular modeling studies, we reveal an unexpected role for the highly conserved transmembrane segment 1 residue Asn-101 in coupling Cl(-) binding to concentrative neurotransmitter uptake.