The Accuracy of Physical Examination to Diagnose Anemia Among Patients Five Years or Older: A Systematic Review.

Anemia remains a significant public health challenge, disproportionately impacting lower-income patients residing in areas of lesser healthcare resources. We sought to evaluate the accuracy of physical exam techniques to diagnose anemia among patients 5 years of age or older. A systematic review of 5 databases (MEDLINE via OVID, EMBASE, Scopus, Global Health and Global Health Archives, and WHO Global Index Medicus) was conducted. Studies that (1) compared non-invasive physical exam techniques with anemia diagnoses using standard laboratory measurements and (2) solely assessed or separately reported the diagnostic accuracy of physical exam techniques for patients 5 years or older were considered for inclusion. The diagnostic accuracies of individual and combinatorial physical exam techniques todiagnose anemia were documented. This systematic review was registered with PROSPERO. The systemic literature search yielded 6,457 unique studies after removal of duplicates. Fourteen studies were ultimately selected for inclusion. Eight studies solely assessed pregnant females, 4 solely assessed hospitalized patients, and 2 evaluated the general population. The diagnostic accuracy ranged widely for pallor assessments of conjunctivae (sensitivity: 19-97%, specificity: 65-100%), nailbed (sensitivity: 41-65%, specificity: 58-93%), and palms (sensitivity: 33-91%, specificity: 54-93%). Examining 9 or more sites leads to higher sensitivity (73.8-82.9%) and specificity (76.0-90.9%). No individual examination technique is superior to others for diagnosing anemia. Combinatorial approachs are associated with more acceptable accuracy measures, but improvements need to be balanced with time available for examination. Supplementary Information: The online version contains supplementary material available at 10.1007/s12288-022-01543-z.

Direct introgression of untapped diversity into elite wheat lines.

The effective utilization of natural variation has become essential in addressing the challenges that climate change and population growth pose to global food security. Currently adopted protracted approaches to introgress exotic alleles into elite cultivars need substantial transformation. Here, through a strategic three-way crossing scheme among diverse exotics and the best historical elites (exotic/elite1//elite2), 2,867 pre-breeding lines were developed, genotyped and screened for multiple agronomic traits in four mega-environments. A meta-genome-wide association study, selective sweeps and haplotype-block-based analyses unveiled selection footprints in the genomes of pre-breeding lines as well as exotic-specific associations with agronomic traits. A simulation with a neutrality assumption demonstrated that many pre-breeding lines had significant exotic contributions despite substantial selection bias towards elite genomes. National breeding programmes worldwide have adopted 95 lines for germplasm enhancement, and 7 additional lines are being advanced in varietal release trials. This study presents a great leap forwards in the mobilization of GenBank variation to the breeding pipelines.

GWAS revealed a novel resistance locus on chromosome 4D for the quarantine disease Karnal bunt in diverse wheat pre-breeding germplasm.

This study was initiated to identify genomic regions conferring resistance to Karnal Bunt (KB) disease in wheat through a genome-wide association study (GWAS) on a set of 179 pre-breeding lines (PBLs). A GWAS of 6,382 high-quality DArTseq SNPs revealed 15 significant SNPs (P-value <10-3) on chromosomes 2D, 3B, 4D and 7B that were associated with KB resistance in individual years. In particular, two SNPs (chromosome 4D) had the maximum R2 values: SNP 1114200 | F | 0-63:T > C at 1.571 cM and R2 of 12.49% and SNP 1103052 | F | 0-61:C > A at 1.574 cM and R2 of 9.02%. These two SNPs displayed strong linkage disequilibrium (LD). An in silico analysis of SNPs on chromosome 4D identified two candidate gene hits, TraesCS4D02G352200 (TaNox8; an NADPH oxidase) and TraesCS4D02G350300 (a rhomboid-like protein belonging to family S54), with SNPs 1103052 | F | 0-61:C > A and 1101835 | F | 0-5:C > A, respectively, both of which function in biotic stress tolerance. The epistatic interaction analysis revealed significant interactions among 4D and 7B loci. A pedigree analysis of confirmed resistant PBLs revealed that Aegilops species is one of the parents and contributed the D genome in these resistant PBLs. These identified lines can be crossed with any elite cultivar across the globe to incorporate novel KB resistance identified on 4B.

Genomic prediction models for grain yield of spring bread wheat in diverse agro-ecological zones.

Genomic and pedigree predictions for grain yield and agronomic traits were carried out using high density molecular data on a set of 803 spring wheat lines that were evaluated in 5 sites characterized by several environmental co-variables. Seven statistical models were tested using two random cross-validations schemes. Two other prediction problems were studied, namely predicting the lines' performance at one site with another (pairwise-site) and at untested sites (leave-one-site-out). Grain yield ranged from 3.7 to 9.0 t ha(-1) across sites. The best predictability was observed when genotypic and pedigree data were included in the models and their interaction with sites and the environmental co-variables. The leave-one-site-out increased average prediction accuracy over pairwise-site for all the traits, specifically from 0.27 to 0.36 for grain yield. Days to anthesis, maturity, and plant height predictions had high heritability and gave the highest accuracy for prediction models. Genomic and pedigree models coupled with environmental co-variables gave high prediction accuracy due to high genetic correlation between sites. This study provides an example of model prediction considering climate data along-with genomic and pedigree information. Such comprehensive models can be used to achieve rapid enhancement of wheat yield enhancement in current and future climate change scenario.

A Comparative Study of the Stress Distribution in Different EndodonticPost-RetainedTeeth with and without Ferrule Design-A Finite Element Analysis.

Purpose. To analyze the stress distribution in an endodontically treated maxillary central incisor restored with various post-core systems and assess the benefit of ferrule using finite element analysis. Material and Methods. Twelve models with metal ceramic crown were created based on the combination of three types of post-core systems (titanium post-composite resin core, nickel-chromium post-core, and fiber reinforced composite resin post-composite resin core), two varieties of posts (tapered, parallel), and with or without ferrule. 100 N load was applied in three directions and the von Mises stress was compared. Results. Ferrule made no difference in stress distribution for the titanium and nickel-chromium posts, though it showed some stress reduction in fiber-reinforced composite resin posts. Nickel-chromium cast post-core transmitted the least amount of stresses to the dentin despite producing the maximum stress. Conclusion. Incorporation of ferrule offered some degree of stress reduction in nonmetal post, and it increased the stresses within cervical dentin.

Developing antibacterial vaccines in genomics and proteomics era.

Infectious diseases are the greatest cause of global morbidity and mortality. About half of this burden is caused by pathogenic bacteria. One of the most effective ways to prevent infectious diseases is to use vaccines. Limitations in the traditional approaches may be one of the reasons why vaccines are yet not available against some infectious bacterial agents. Recent advancements in high-throughput 'omics' technologies and the availability of complete genome sequences of microbial pathogens and multiple isolates of the same species have dramatically changed the time frame and scope for identifying novel vaccine candidates. At present, 582 microbial genomes have been sequenced and sequencing of 1733 is in progress. There are more than 50 bacterial species for which the genome sequence is available for three or more isolates. Development of tools for in silico analysis has led to the identification of novel virulence genes, metabolic pathways and cell surface proteins that represent potential new targets for anti-microbial drug and vaccine strategies. In this review, we provide an overview of the 'omics'-based techniques that can be used to advance and accelerate the discovery of vaccine candidates against extracellular bacterial pathogens. By citing specific examples, we discuss how high-throughput molecular profiling techniques, such as genomics, transcriptomics and proteomics have contributed to the discovery of novel vaccine candidates. We end by contemplating on the emerging technologies that are likely to have a high impact on the field of vaccinology in the near future.

The Kit-activating mutation D816V enhances stem cell factor--dependent chemotaxis.

The D816V mutation of c-kit has been detected in patients with mastocytosis. This mutation leads to constitutive tyrosine kinase activation of Kit. Because stem cell factor (SCF), the ligand for Kit (CD117(+)), is a chemoattractant for CD117(+) cells and one feature of mastocytosis is an abnormal collection of mast cells in tissues derived from CD34(+)CD117(+) mast cell precursors, the hypothesis was considered that the D816V mutation would enhance chemotaxis of these precursor cells. Constructs encoding wild-type Kit or Kit bearing the D816V mutation were transfected into Jurkat cells, labeled with Calcein-AM, and migration to SCF assessed in the presence or absence of tyrosine kinase inhibitors. Chemotaxis to SCF was enhanced in D816V transfectants compared to wild-type Kit transfectants (P <.002). Migration of both transfectants was inhibited by tyrosine kinase inhibitors, although D816V transfectants were more sensitive. Chemotaxis was next performed on CD34(+)CD117(+) circulating mast cell precursors obtained from patients with mastocytosis. Analysis of prechemotaxis and migrated cells showed that whereas less than 10% in the prechemotaxis sample had the D816V mutation, 40% to 80% of migrated cells had this mutation. These results demonstrate that the D816V Kit mutation enhances chemotaxis of CD117(+) cells, offering one explanation for increased mast cells observed in tissues of patients with mastocytosis. (Blood. 2001;98:1195-1199)

The ORF3 protein of hepatitis E virus binds to Src homology 3 domains and activates MAPK.

The hepatitis E virus (HEV) is the causative agent of hepatitis E, an acute form of viral hepatitis. The biology and pathogenesis of HEV remain poorly understood. We have used in vitro binding assays to show that the HEV ORF3 protein (pORF3) binds to a number of cellular signal transduction pathway proteins. This includes the protein tyrosine kinases Src, Hck, and Fyn, the p85alpha regulatory subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma, and the adaptor protein Grb2. A yeast two-hybrid assay was used to further confirm the pORF3-Grb2 interaction. The binding involves a proline-rich region in pORF3 and the src homology 3 (SH3) domains in the cellular proteins. Competition assays and computer-assisted modeling was used to evaluate the binding surfaces and interaction energies of the pORF3.SH3 complex. In pORF3-expressing cells, pp60(src) was found to associate with an 80-kDa protein, but no activation of the Src kinase was observed in these cells. However, there was increased activity and nuclear localization of ERK in the pORF3-expressing cells. These studies suggest that pORF3 is a viral regulatory protein involved in the modulation of cell signaling. The ORF3 protein of HEV appears to be the first example of a SH3 domain-binding protein encoded by a virus that causes an acute and primarily self-limited infection.

Generation of heterogeneous rabbit anti-DNP antibodies by gene conversion and hypermutation of rearranged VL and VH genes during clonal expansion of B cells in splenic germinal centers.

The mechanisms described here account for development of the heterogeneous high-affinity anti-DNP antibodies that rabbits can produce. Rearranged immunoglobulin light and heavy chain genes from single DNP-specific splenic germinal center B cells were amplified by PCR. We found that in clonal lineages, rearranged V[kappa] and V[H] are further diversified by gene conversion and somatic hypermutation. The positive and negative selection of amino acids in complementarity-determining regions observed allows emergence of a variety of different combining site structures. A by-product of the germinal center reaction may be cells with sequences altered by gene conversion that no longer react with the immunizing antigen but are a source of new repertoire. The splenic germinal center would thus play an additional role in adults similar to that of the appendix and other gut-associated lymphoid tissues of young rabbits.

Gene-conversion in rabbit B-cell ontogeny and during immune responses in splenic germinal centers.

Combinatorial diversity is limited in rabbits because only a few V(H) genes rearrange. Most diversification of the primary repertoire is generated by somatic hypermutation and gene conversion-like changes of rearranged V(H) in B cells that migrate to appendix and other gut associated lymphoid tissues (GALT) of young rabbits. The changes are referred to as gene conversion-like because the non-reciprocal nature of the alterations introduced has not yet been demonstrated. There are many similarities between rabbits and chickens in how their B cells develop and diversify their repertoires. However, although the majority of rabbit B cells may have rearranged and diversified their V genes early in life, some B cells in adult rabbits have rearranged VH sequences that are identical or nearly identical to germline sequences. We found these cells in splenic germinal centers (GC) on days 7 and 10 after immunization of normal adult rabbits with DNP-BGG. By day 15, all rearranged V(H) sequences were diversified. We find an overall pattern of splenic precursor cells whose germline or near germline sequences change both by gene conversion and point mutations during early divisions and mainly by point mutations during later divisions. These events, in parallel with diversification of light chain sequences, may produce the diverse combining sites that serve as substrates for further affinity maturation by selection either within GC or later among emigrant cells in sites such as bone marrow. Some of the sequences altered by gene conversion in splenic germinal centers may also produce new members of the B-cell repertoire in adult rabbits comparable to those produced in GALT of neonatal rabbits.

Generation of the primary antibody repertoire in rabbits: expression of a diverse set of Igk-V genes may compensate for limited combinatorial diversity at the heavy chain locus.

In mouse and human, generation of combinatorial diversity through use of different heavy and light chain variable region genes in immunoglobulin rearrangements can be a major contributor to the primary antibody repertoire. In rabbits, the contribution of the combinatorial mechanism to heavy chain diversity is minimal, as only a few Igh-V genes are rearranged and expressed. To investigate the contribution of combinatorial diversity toward generation of the rabbit V(kappa) repertoire, we constructed five genomic libraries from rabbit kidney DNA and 1 cDNA library from the bone marrow of a 1-day-old rabbit using a series of polymerase chain reaction-based strategies. Our analyses indicate that most of the sequences that we recovered from our libraries belong to a single family and some are extremely similar. The actual number of germline Igk-V genes is potentially greater than our conservative estimate of at least 39, 28 of which we found expressed as mRNA. The germline Igk-V genes display different lengths of the coding region 3' of Cys 88 ranging from 7 to 12 amino acids, resulting in CDR3 length heterogeneity among functional V(kappa)J(kappa) sequences ranging from 8 to 15 amino acids. Some of the V(kappa)J(kappa) junctions had N and P nucleotide additions. Thus, in contrast to limited combinatorial diversity of its heavy chain, the rabbit can draw upon a diverse set of germline Igk-V genes. The kappa light chain has the potential to be a major contributor toward generation of the antibody specificities of the rabbit pre-immune repertoire.

Gene conversion and hypermutation during diversification of VH sequences in developing splenic germinal centers of immunized rabbits.

The young rabbit appendix and the chicken bursa of Fabricius are primary lymphoid organs where the B cell Ab repertoire develops in germinal centers (GCs) mainly by a gene conversion-like process. In human and mouse, V-gene diversification by somatic hypermutation in GCs of secondary lymphoid organs leads to affinity maturation. We asked whether gene conversion, somatic hypermutation, or both occur in rabbit splenic GCs during responses to the hapten DNP. We determined DNA sequences of rearranged heavy and light chain V region gene segments in single cells from developing DNP-specific GCs after immunization with DNP-bovine gamma-globulin and conclude that the changes at the DNA level that may lead to affinity maturation occur by both gene conversion and hypermutation. Selection was suggested by finding some recurrent amino acid replacements that may contribute increased affinity for antigen in the complementarity-determining region sequences of independently evolved clones, and a narrower range of complementarity-determining region 3 lengths at day 15. Some of the alterations of sequence may also lead to new members of the B cell repertoire in adult rabbits comparable with those produced in gut associated lymphoid tissues of young rabbits.

Recombinant Bombyx mori nucleopolyhedrovirus harboring green fluorescent protein.

The gene encoding the green fluorescent protein (gfp) under the control of the highly expressed Autographa californica nucleopolyhedrovirus (AcMNPV)-polyhedrin promoter has been introduced into the polyhedrin (polh) locus of Bombyx mori nucleopolyhedrovirus (BmNPV) by homologous recombination. The insect host larvae and the cultured cells infected with this recombinant virus (vBmGFP) showed high levels of expression of gfp. The larval tissues permissive to virus multiplication could be readily visualized using the tagged recombinant virus, thus providing a direct approach to study the progress of virus infection or its control in the animal host. The highly expressed recombinant protein, GFP, could be easily solubilized from fat bodies. Thus, the caterpillar-based expression could serve as an economic alternative method for the large-scale production of recombinant proteins, even when they are nonsecretory in nature. Further, if the recombinant vBmGFP is used as a parent in generating other recombinants, conversion of the fluorescent plaques to colorless plaques serves as an easy means for screening recombinants. Such a method is especially helpful for BmNPV-recombinant selections in the absence of the other simplified techniques as are available for the prototype baculovirus AcMNPV system.

Analyses of single B cells by polymerase chain reaction reveal rearranged VH with germline sequences in spleens of immunized adult rabbits: implications for B cell repertoire maintenance and renewal.

We used PCR to amplify rearranged VHDJH genes in single cells collected by micromanipulation from splenic germinal centers of immunized adult rabbits. In the course of the study, the objective of which was to analyze diversification of rearranged VHDJH sequences, we were surprised to find cells 7 and 10 days after immunization with rearranged VH1a2 as well as a-negative (y33 and x32) sequences that were identical or close to germline (10 or fewer changes). About 58% (82/140) of the sequences had unique CDR3 regions and were unrelated. In seven different germinal centers, we found one to four different clones with two to seven members. Clonally related cells underwent diversification by hypermutation and gene conversion. We found that contrary to published reports, adult rabbits indeed have newly diversifying B cell receptors in splenic germinal centers. The attractive idea that the rabbit, like the chicken, develops its B cell repertoire early in life and depends upon self-renewing cells in the periphery to maintain its B lymphocyte pool throughout life, is challenged by the current finding. Although a major population of B lymphocytes may be generated early in life, diversified extensively, and maintained by self-renewal in the periphery, some sources of cells with sequences close to germline do exist in adult rabbits and appear in the developing germinal centers. Although considerable repertoire diversity is generated in young rabbits, mechanisms for continued generation of B cell receptor diversity are retained in adult life, where they may confer survival advantage.

VH mutant rabbits lacking the VH1a2 gene develop a2+ B cells in the appendix by gene conversion-like alteration of a rearranged VH4 gene.

We investigated the molecular basis for the appearance of V(H)a2 allotype-bearing B cells in mutant Alicia rabbits. The mutation arose in an a2 rabbit; mutants exhibit altered expression of V(H) genes because of a small deletion encompassing V(H)1a2, the 3'-most gene in the V(H) locus. The V(H)1 gene is the major source of V(H)a allotype because this gene is preferentially rearranged in normal rabbits. In young homozygous ali/ali animals, the levels of a2 molecules found in the serum increase with age. In adult ali/ali rabbits, 20 to 50% of serum Igs and B cells bear a2 allotypic determinants. Previous studies suggested that positive selection results in expansion of a2 allotype-bearing B cells in the appendix of young mutant ali/ali rabbits. We separated appendix cells from a 6-wk-old Alicia rabbit by FACS based on the expression of surface IgM and a2 allotype. The VDJ portion of the expressed Ig mRNA was amplified from the IgM+ a2+ and IgM+ a2- populations by reverse transcriptase-PCR. The cDNAs from both populations were cloned and sequenced. Analysis of these sequences suggested that, in a2+ B cells, the first D proximal functional gene in Alicia rabbits, V(H)4a2, rearranged and was altered further by a gene conversion-like mechanism. Upstream V(H) genes were identified as potential gene sequence donors; V(H)9 was found to be the most frequently used gene donor. Among the a2- B cells, y33 was the most frequently rearranged gene.

Identification of novel genes from Entamoeba histolytica by expressed sequence tag analysis.

Shotgun sequencing of cDNA clones is now an established approach to gain insight into the expressed nucleotide (nt) sequences in a given cell. We analysed 100 randomly picked cDNA clones of the protozoan parasite, Entamoeba histolytica, by nt sequencing, with a view to obtain novel gene sequences not detected so far by biochemical and genetic analyses. About 56% of the analysed clones showed significant homology with other genes in the database, including a number of genes whose presence may not be suspected in E. histolytica owing to its unusual subcellular organization. The results suggest that this approach can provide important clues to understand unique biochemical mechanisms in this parasite.

Nucleotide sequence organisation and analysis of the nuclear ribosomal DNA circle of the protozoan parasite Entamoeba histolytica.

We have sequenced the extrachromsomal ribosomal DNA (rDNA) circle of the human protozoan parasite Entamoeba histolytica HM-1:IMSS and present here the complete sequence organisation of the 24.5-kb molecule. Each circle contains two 5.9-kb rDNA transcription units organised as inverted repeats. The regions downstream (3543 bp) and upstream (9216 bp) of the rDNAs contain various families of short tandem repeats. Some of the upstream repeats share extensive sequence homology with the downstream repeats. In addition to the rDNAs themselves, the rDNA circle appears to code for only one other transcript which is 0.7 kb in size as seen in Northern blots. From DNA sequence analysis, no open reading frame could be assigned to the transcript. Extrachromosomal rDNA circles also exist in other E. histolytica strains. Restriction enzyme maps of rDNA circles were constructed from E. histolytica strains 200:NIH, HK-9 and Rahman; and Entamoeba moshkovskii strain Laredo. Striking differences were observed in the organisation of some of them, e.g. the HK-9, Rahman and Laredo circles contained only one rDNA unit and lacked the 0.7-kb transcript sequence. The short repeat sequences upstream and downstream of rDNAs were present in HK-9 and Rahman but absent in Laredo. Circles with one rDNA unit may be derived from those with two units by homologous recombination at direct repeat sequences located upstream and downstream of the two rDNAs.

A method for the high efficiency of water-soluble carbodiimide-mediated amidation.

Water-soluble carbodiimides are widely used for carboxyl-amine conjugation. However, extremely variable and low yields, obtained under a variety of conditions, have been a serious problem in the coupling. A simple method, optimizing various parameters of the coupling reaction, in which N-hydroxysuccinimide is included to assist the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride-catalyzed amidation reaction is described. A product yield of up to 90% is routinely achieved.