Conventional kinesin holoenzymes are composed of heavy and light chain homodimers

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.     

Genome-wide haploinsufficiency screen reveals a novel role for γ-TuSC in spindle organization and genome stability

How subunit dosage contributes to the assembly and function of multimeric complexes is an important question with implications in understanding biochemical, evolutionary, and disease mechanisms. Toward identifying pathways that are susceptible to decreased gene dosage, we performed a genome-wide screen for haploinsufficient (HI) genes that guard against genome instability in Saccharomyces cerevisiae. This led to the identification of all three genes (SPC97, SPC98, and TUB4) encoding the evolutionarily conserved γ-tubulin small complex (γ-TuSC), which nucleates microtubule assembly. We found that hemizygous γ-TuSC mutants exhibit higher rates of chromosome loss and increases in anaphase spindle length and elongation velocities. Fluorescence microscopy, fluorescence recovery after photobleaching, electron tomography, and model convolution simulation of spc98/+ mutants revealed improper regulation of interpolar (iMT) and kinetochore (kMT) microtubules in anaphase. The underlying cause is likely due to reduced levels of Tub4, as overexpression of TUB4 suppressed the spindle and chromosome segregation defects in spc98/+ mutants. We propose that γ-TuSC is crucial for balanced assembly between iMTs and kMTs for spindle organization and accurate chromosome segregation. Taken together, the results show how gene dosage studies provide critical insights into the assembly and function of multisubunit complexes that may not be revealed by using traditional studies with haploid gene deletion or conditional alleles.     

Acute psychiatric care: approaches to increasing the range of services and improving access and quality of care

Acute services for mental health crises are very important to service users and their supporters, and consume a substantial share of mental health resources in many countries. However, acute care is often unpopular and sometimes coercive, and the evidence on which models are best for patient experience and outcomes remains surprisingly limited, in part reflecting challenges in conducting studies with people in crisis. Evidence on best ap­proaches to initial assessment and immediate management is particularly lacking, but some innovative models involving extended assessment, brief interventions, and diversifying settings and strategies for providing support are potentially helpful. Acute wards continue to be central in the intensive treatment phase following a crisis, but new approaches need to be developed, evaluated and implemented to reducing coercion, addressing trauma, diversifying treatments and the inpatient workforce, and making decision‐making and care collaborative. Intensive home treatment services, acute day units, and community crisis services have supporting evidence in diverting some service users from hospital admission: a greater understanding of how best to implement them in a wide range of contexts and what works best for which service users would be valuable. Approaches to crisis management in the voluntary sector are more flexible and informal: such services have potential to complement and provide valuable learning for statutory sector services, especially for groups who tend to be underserved or disengaged. Such approaches often involve staff with personal experience of mental health crises, who have important potential roles in improving quality of acute care across sectors. Large gaps exist in many low‐ and middle‐income countries, fuelled by poor access to quality mental health care. Responses need to build on a foundation of existing community responses and contextually relevant evidence. The necessity of moving outside formal systems in low‐resource settings may lead to wider learning from locally embedded strategies.     

Heterogeneity of ADP diffusion and regulation of respiration in cardiac cells

Heterogeneity of ADP diffusion and regulation of respiration were studied in permeabilized cardiomyocytes and cardiac fibers in situ and in silico. Regular arrangement of mitochondria in cells was altered by short-time treatment with trypsin and visualized by confocal microscopy. Manipulation of matrix volumes by changing K(+) and sucrose concentrations did not affect the affinity for ADP either in isolated heart mitochondria or in skinned fibers. Pyruvate kinase (PK)-phosphoenolpyruvate (PEP) were used to trap ADP generated in Ca,MgATPase reactions. Inhibition of respiration by PK-PEP increased 2-3 times after disorganization of regular mitochondrial arrangement in cells. ADP produced locally in the mitochondrial creatine kinase reaction was not accessible to PK-PEP in intact permeabilized fibers, but some part of it was released from mitochondria after short proteolysis due to increased permeability of outer mitochondrial membrane. In in silico studies we show by mathematical modeling that these results can be explained by heterogeneity of ADP diffusion due to its restrictions at the outer mitochondrial membrane and in close areas, which is changed after proteolysis. Localized restrictions and heterogeneity of ADP diffusion demonstrate the importance of mitochondrial functional complexes with sarcoplasmic reticulum and myofibrillar structures and creatine kinase in regulation of oxidative phosphorylation.     

Development and characterization of a compensating wheat-Thinopyrum intermedium Robertsonian translocation with Sr44 resistance to stem rust (Ug99)

The emergence of the highly virulent Ug99 race complex of the stem rust fungus (Puccinia graminis Pers. f. sp. tritici Eriks. and Henn.) threatens wheat (Triticum aestivum L.) production worldwide. One of the effective genes against the Ug99 race complex is Sr44, which was derived from Thinopyrum intermedium (Host) Barkworth and D.R. Dewey and mapped to the short arm of 7J (designated 7J#1S) present in the noncompensating T7DS-7J#1L?7J#1S translocation. Noncompensating wheat-alien translocations are known to cause genomic duplications and deficiencies leading to poor agronomic performance, precluding their direct use in wheat improvement. The present study was initiated to produce compensating wheat-Th. intermedium Robertsonian translocations with Sr44 resistance. One compensating RobT was identified consisting of the wheat 7DL arm translocated to the Th. intermedium 7J#1S arm resulting in T7DL?7J#1S. The T7DL?7J#1S stock was designated as TA5657. The 7DL?7J#1S stock carries Sr44 and has resistance to the Ug99 race complex. This compensating RobT with Sr44 resistance may be useful in wheat improvement. In addition, we identified an unnamed stem rust resistance gene located on the 7J#1L arm that confers resistance not only to Ug99, but also to race TRTTF, which is virulent to Sr44. However, the action of the second gene can be modified by the presence of suppressors in the recipient wheat cultivars.     

Homogeneously sized groups increase aggressive interaction and affect social stress in Thai strain Nile tilapia (Oreochromis niloticus)

Social fish raised in farms are usually kept in groups of similar-sized individuals. However, social animals of similar size typically have similar fighting ability, which increases aggressive interaction for social rank establishment, as well as social stress. We compared Thai strain Nile tilapia fish, Oreochromis niloticus (L.), held under two treatments: (1) The Homogeneous one, with five adult male fish of similar size and (2) the Heterogeneous treatment with five adult males of different sizes. We recorded the frequency of aggressive interactions and checked social stability and stress levels (cortisol) after five days in the groups. Grouping similar sized Thai Nile tilapia increased the aggressive interactions and delayed rank stability with increased body injuries as a consequence. Homogeneous-sized individuals showed a similar level of stress while heterogeneous-sized individuals showed different stress levels with dominants being more stressed than subordinates. The data indicate that the practice of selecting fish of similar size in aquaculture management could reduce the welfare of social fish and that the effect is observed in different lineages.     

Synthesis of oligonucleotide 2'-conjugates via amide bond formation in solution

An efficient method for synthesis of 2'-O-carboxymethyl oligonucleotides is described. Fully deprotected oligonucleotides containing a carboxymethyl group at the 2'-position of sugar residue were obtained by a two-step procedure by periodate cleavage of an oligonucleotide containing 1,2-diol group followed by oxidation of the 2'-aldehyde resulted with sodium chlorite. 2'-O-Carboxymethyl oligonucleotides prepared were efficiently coupled in aqueous solution in the presence of a water-soluble carbodiimide to a number of amino acid derivatives or short peptides to afford novel 2'-conjugates of high purity in good yield. The method is thus shown to be suitable in principle for preparation of oligonucleotide-peptide conjugates containing an amide linkage between the 2'-carboxy group of a modified oligonucleotide and the amino terminus of a peptide.