Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Immobilization-dependent bone collagen breakdown appears to increase with time: evidence for a lack of new bone equilibrium in response to reduced load during prolonged bed rest.

The purpose of this study was to evaluate the effect of prolonged immobilization on bone, in order to investigate how skeletal turnover adapts to bed rest. We examined indices of bone formation and bone resorption in the serum and urine of fifty-four patients (26 males and 28 females) immobilized after an episode of paralytic stroke. The length of immobilization ranged from 30 to 180 days. A significant, time-dependent increase in markers of resorption - urinary pyridinoline (Pyr) and deoxypyridinoline (D-Pyr), serum Type I collagen cross-linked C-telopeptide (ICTP) - was observed in immobilized patients, as compared to free-living healthy subjects. The positive correlation between resorption markers increase and the length of immobilization suggests that the rate of bone resorption did not decrease with time. On the other hand, the levels of markers of bone formation - bone-specific alkaline phosphatase (B-ALP), and the carboxyl-terminal propeptide of Type I procollagen (PICP) - remained within the normal range in all patients, regardless the length of immobilization. Our results would indicate an uncoupling between bone formation and bone resorption during bed rest, and suggest that the bone collagen break-down was not a self-limiting process in immobilized patients, and that a new equilibrium or "steady state" in response to the reduced load was not reached in the skeleton.     

Seasonal suitability of three rubber tree clones to Calacarus heveae (Acari, Eriophyidae)

The suitability of rubber tree clones to Calacarus heveae was inferred from the life cycle, reproduction and survivorship of this mite. The assays were performed under controlled conditions with leaflets detached from 6-year-old plants. The development of 20 C. heveae individuals on each of the clones GT 1, PB 235 and RRIM 600 was analysed. This experiment was performed four times during periods when C. heveae was abundant in the field: (P1) November–December 2005, (P2) January–February, (P3) March–April and (P4) May–June 2006. Accordingly, the leaflets used in each assay represented the physiological condition of the host plant during each period. This approach allowed us to evaluate the seasonal suitability of rubber tree clones to C. heveae. We observed seasonal differences in the suitability of rubber tree clones to mite attack. The mites reared on the PB235 had a shorter development period, the highest egg production and highest survivorship. This evidence showed that the PB 235 was the most suitable of those tested. We also observed that the leaflets used in the assays during periods P2 and P3 were the most favourable for the development of C. heveae. This finding emphasises the seasonal suitability of rubber tree leaflets. On the other hand, GT 1 showed higher resistance against C. heveae than did RRIM 600 and PB 235, primarily during the period from November to February. This result indicated that use of the GT 1 clone to control the mite might represent an alternative for growers.     

Condensin complexes regulate mitotic progression and interphase chromatin structure in embryonic stem cells

In an RNA interference screen interrogating regulators of mouse embryonic stem (ES) cell chromatin structure, we previously identified 62 genes required for ES cell viability. Among these 62 genes were Smc2 and -4, which are core components of the two mammalian condensin complexes. In this study, we show that for Smc2 and -4, as well as an additional 49 of the 62 genes, knockdown (KD) in somatic cells had minimal effects on proliferation or viability. Upon KD, Smc2 and -4 exhibited two phenotypes that were unique to ES cells and unique among the ES cell–lethal targets: metaphase arrest and greatly enlarged interphase nuclei. Nuclear enlargement in condensin KD ES cells was caused by a defect in chromatin compaction rather than changes in DNA content. The altered compaction coincided with alterations in the abundance of several epigenetic modifications. These data reveal a unique role for condensin complexes in interphase chromatin compaction in ES cells.     

NmeCas9 is an intrinsically high-fidelity genome-editing platform

Background

The development of CRISPR genome editing has transformed biomedical research. Most applications reported thus far rely upon the Cas9 protein from Streptococcus pyogenes SF370 (SpyCas9). With many RNA guides, wildtype SpyCas9 can induce significant levels of unintended mutations at near-cognate sites, necessitating substantial efforts toward the development of strategies to minimize off-target activity. Although the genome-editing potential of thousands of other Cas9 orthologs remains largely untapped, it is not known how many will require similarly extensive engineering to achieve single-site accuracy within large genomes. In addition to its off-targeting propensity, SpyCas9 is encoded by a relatively large open reading frame, limiting its utility in applications that require size-restricted delivery strategies such as adeno-associated virus vectors. In contrast, some genome-editing-validated Cas9 orthologs are considerably smaller and therefore better suited for viral delivery.

Results

Here we show that wildtype NmeCas9, when programmed with guide sequences of the natural length of 24 nucleotides, exhibits a nearly complete absence of unintended editing in human cells, even when targeting sites that are prone to off-target activity with wildtype SpyCas9. We also validate at least six variant protospacer adjacent motifs (PAMs), in addition to the preferred consensus PAM (5′-N₄GATT-3′), for NmeCas9 genome editing in human cells.

Conclusions

Our results show that NmeCas9 is a naturally high-fidelity genome-editing enzyme and suggest that additional Cas9 orthologs may prove to exhibit similarly high accuracy, even without extensive engineering.

     

Assessment of Cu-Zn EDTA Parenteral Toxicity in Calves

Copper (Cu) parenteral administration is used in a beef cow-calf operations to prevent or correct Cu deficiency in bovines. At present, Zinc (Zn) salts have been incorporated to complement Cu antioxidant effect. A risk of hepatotoxicity generated by overdose is a negative consequence of injectable Cu application. Cu-Zn EDTA appears as an alternative; however, data about its toxicity is unknown. The aim of this study was to assess toxicity risk of different doses of Cu-Zn EDTA in calves. Thirty two Aberdeen Angus calves of 162 (±20) kg BW were assigned to 4 groups (n = 8), homogeneous in weight, sex, and age. Cu-Zn EDTA was administrated in doses of 0.3 mg/kg BW (group 1X); 0.6 mg/kg BW (group 2X); 0.9 mg/kg BW (group 3X) and sterile saline solution (control group-with no treatment). Clinical and blood parameters in animals were monitored during 28 days. In groups’ control, 1X and 2X there were no alterations in the assessed parameters. In group 3X, one of the animals showed depression, permanent decubitus, and muscular twitching; that animal had to be killed in extremis for humanitarian reasons. Necropsy and Cu tissue concentration findings confirmed intoxication in the clinically affected animal. The rest of the animals in group 3X showed only a temporary increase in liver enzymes. The results indicate that a dose of 0.9 mg/kg BW of Cu as Cu-Zn EDTA is potentially hepatotoxic, this dose is similar to other soluble salts of parenteral administration.     

Unbiased chromatin accessibility profiling by RED-seq uncovers unique features of nucleosome variants in vivo

Background

Differential accessibility of DNA to nuclear proteins underlies the regulation of numerous cellular processes. Although DNA accessibility is primarily determined by the presence or absence of nucleosomes, differences in nucleosome composition or dynamics may also regulate accessibility. Methods for mapping nucleosome positions and occupancies genome-wide (MNase-seq) have uncovered the nucleosome landscapes of many different cell types and organisms. Conversely, methods specialized for the detection of large nucleosome-free regions of chromatin (DNase-seq, FAIRE-seq) have uncovered numerous gene regulatory elements. However, these methods are less successful in measuring the accessibility of DNA sequences within nucelosome arrays.

Results

Here we probe the genome-wide accessibility of multiple cell types in an unbiased manner using restriction endonuclease digestion of chromatin coupled to deep sequencing (RED-seq). Using this method, we identified differences in chromatin accessibility between populations of cells, not only in nucleosome-depleted regions of the genome (e.g., enhancers and promoters), but also within the majority of the genome that is packaged into nucleosome arrays. Furthermore, we identified both large differences in chromatin accessibility in distinct cell lineages and subtle but significant changes during differentiation of mouse embryonic stem cells (ESCs). Most significantly, using RED-seq, we identified differences in accessibility among nucleosomes harboring well-studied histone variants, and show that these differences depend on factors required for their deposition.

Conclusions

Using an unbiased method to probe chromatin accessibility genome-wide, we uncover unique features of chromatin structure that are not observed using more widely-utilized methods. We demonstrate that different types of nucleosomes within mammalian cells exhibit different degrees of accessibility. These findings provide significant insight into the regulation of DNA accessibility.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1104) contains supplementary material, which is available to authorized users.