Improving child survival under National Health Mission in India: Where do we stand?

India contributes to around one-fifth of the global under-five mortality and also maternal mortality besides one-third of the neonatalmortality. Since any reduction in child mortality in India is crucial for the global decline, therefore, the Indian Government decidedto undertake massive correction of the health system. This led to the launch of National Rural Health Mission in the year 2005. Sincethen, significant progress has been made and child mortality rates have shown a sharp decline. On comparing the progress made by theworld toward Millennium Development Goals, India fares better by showing a decline of 46.5% in comparison to 41% for the entireworld during the same period. In order to assess the state-wise reduction, data from sample registration system of the Registrar Generalof India which is available for most of the States/Union Territories (UTs) have been analyzed. States such as Maharashtra, Tamil Nadu,and Kerala have shown an impressive decline but some states such as Himachal Pradesh, Punjab, Mizoram, and Delhi still have a longway to go to reach the state specific goals and targets. Any further decline would only be possible by addressing inter-district variationsthat are still lagging behind and focused efforts need to be made, in order to reach these desired goals. This analysis would be valuablein planning future program implementation plans

Procjena kakvoće ploda novog hibrida pomela i slatke naranče te njegova molekularna karakterizacija pomoću genetskih biljega specifičnih za kiselost

Research backgroundThere is considerable diversity in newly developed pummelo × sweet orange citrus hybrids. Most hybrids showed lower peel thickness and high juice yield but there is a lack of information on fruit quality parameters and molecular characterization. Therefore, the aim of the current study is to determine the content of antioxidants and properties of the fresh juice of 24 new pummelo × sweet orange citrus hybrids (Citrus maxima [Burm. f.] Osbeck × Citrus sinensis [L.] Osbeck) and the parental genotypes along with molecular characteristics determined using acidity specific markers. Experimental approach. The correlation and estimate of inheritance of the fruit juice properties: ascorbic acid, total phenol, total flavonoid, total antioxidant, total soluble solid and sugar contents, pH, titratable acidity, along with sensory evaluation was performed. Molecular characterization of these hybrids was carried out using de novo generated acidity specific simple sequence repeat (SSR) markers. Results and conclusions. The main constituents of the fruit juice of pummelo × sweet orange hybrids were observed in the range of w(ascorbic acid)=40.00–58.13 mg/100 g, total phenols expressed as gallic acid equivalents w(GAE)=40.67–107.33 mg/100 g, total antioxidants expressed as Trolox equivalents b(Trolox)=2.03–5.49 µmol/g, total flavonoids expressed as quercetin equivalents w(QE)=23.67–59.33 mg/100 g, along with other properties: total soluble solids=7.33–11.33 %, w(total sugar)=2.10–5.76 %, w(reducing sugar)=1.69–2.78 %, w(non-reducing sugar)=0.39–3.17 % and titratable acidity 1.00–2.11 %. The above parameters differed significantly in the fruit juice of the evaluated pummelo × sweet orange hybrids. Considering these parameters, the hybrids SCSH 17-9, SCSH 13-13, SCSH 11-15 and SCSH 3-15 had superior antioxidant properties in terms of these parameters. A higher heritability (≥80 %) was also observed for all juice properties. Molecular characterization of pummelo × sweet orange hybrids showed that >50 % of the hybrids were grouped with medium acidity parents. Both molecular and biochemical parameter-based clustering showed that interspecific hybrids exhibit transgressive segregation with increased antioxidants that help alleviate the health problems. Novelty and scientific contribution. These newly developed pummelo × sweet orange citrus hybrids are a valuable source of high-quality antioxidants for a healthy diet. The identification of trait markers that enable selection at the seedling stage is of great benefit to citrus breeders, as the characteristic features of a mature tree are not yet visible at the juvenile stage.Pozadina istraživanja. Među novorazvijenim citrusnim hibridima pomela i slatke naranče postoji bitna različitost. Većina ih ima tanju koru i veliki prinos soka, no dosad nije bilo podataka o parametrima kakvoće ploda i njihovoj molekularnoj karakterizaciji. Stoga je svrha ovog istraživanja bila odrediti udjel antioksidansa i svojstva svježeg soka 24 novih citrusnih hibrida pomela i slatke naranče (Citrus maxima [Burm. f.] Osbeck × Citrus sinensis [L.] Osbeck), te roditeljske genotipove zajedno s molekularnim značajkama određenim pomoću genetskih biljega specifičnih za kiselost. Eksperimentalni pristup. Utvrđene su korelacije i procijenjeno je nasljeđivanje sljedećih svojstava voćnog soka: maseni udjeli askorbinske kiseline, ukupnih fenola, flavonoida, antioksidansa, topljivih tvari i šećera, te pH-vrijednost, titracijska kiselost, kao i senzorska svojstva. Molekularna karakterizacija ovih hibrida provedena je korištenjem de novo generiranih mikrosatelitnih biljega (jednostavnih ponavljajućih sekvencija (SSR)) specifičnih za kiselost. Rezultati i zaključci. Glavni sastojci voćnog soka hibrida pomela i slatke naranče bili su u rasponu w(askorbinska kiselina)=40,00–58,13 mg/100 g, ukupni fenoli izraženi kao ekvivalenti galne kiseline w(GAE)=40,67–107,33 mg/100 g, ukupni antioksidansi izraženi kao ekvivalenti Troloksa b(Troloks)=2,03–5,49 µmol/g, ukupni flavonoidi izraženi kao ekvivalenti kvercetina w(QE)=23,67–59,33 mg/100 g, zajedno s drugim svojstvima: ukupna topljiva čvrsta tvar TSS=7,33 –11,33 %, w(ukupni šećer)=2,10–5,76 %, w(reducirajući šećer)=1,69–2,78 %, w(nereducirajući šećer)=0,39–3,17 % i titracijska kiselost 1,00–2,11 %. Navedeni parametri bitno su se razlikovali u voćnom soku ocjenjivanih hibrida pomela i slatke naranče. S obzirom na ove parametre, hibridi SCSH 17-9, SCSH 13-13, SCSH 11-15 i SCSH 3-15 imali su superiorna antioksidacijska svojstva. Također je uočena veća nasljednost (≥80 %) svih svojstava soka. Molekularna karakterizacija hibrida pomela i slatke naranče pokazala je da je >50 % hibrida grupirano s roditeljima koji imaju srednju kiselost. Grupiranje prema molekularnim i biokemijskim parametrima pokazalo je da interspecifični hibridi pokazuju transgresivnu segregaciju s povećanim udjelom antioksidansa, koji pomažu pri ublažavanju zdravstvenih poteškoća. Novina i znanstveni doprinos. Ovi novorazvijeni citrusni hibridi pomela i slatke naranče vrijedan su izvor visokokvalitetnih antioksidansa za zdravu prehranu. Identifikacija biljega svojstava koji omogućuju selekciju biljke u fazi sadnice od velike je koristi za uzgajivače agruma, jer karakteristična obilježja zrelog stabla još nisu vidljiva u fazi mlade biljke

A novel helper phage enabling construction of genome-scale ORF-enriched phage display libraries.

Phagemid-based expression of cloned genes fused to the gIIIP coding sequence and rescue using helper phages, such as VCSM13, has been used extensively for constructing large antibody phage display libraries. However, for randomly primed cDNA and gene fragment libraries, this system encounters reading frame problems wherein only one of 18 phages display the translated foreign peptide/protein fused to phagemid-encoded gIIIP. The elimination of phages carrying out-of-frame inserts is vital in order to improve the quality of phage display libraries. In this study, we designed a novel helper phage, AGM13, which carries trypsin-sensitive sites within the linker regions of gIIIP. This renders the phage highly sensitive to trypsin digestion, which abolishes its infectivity. For open reading frame (ORF) selection, the phagemid-borne phages are rescued using AGM13, so that clones with in-frame inserts express fusion proteins with phagemid-encoded trypsin-resistant gIIIP, which becomes incorporated into the phages along with a few copies of AGM13-encoded trypsin-sensitive gIIIP. In contrast, clones with out-of-frame inserts produce phages carrying only AGM13-encoded trypsin-sensitive gIIIP. Trypsin treatment of the phage population renders the phages with out-of-frame inserts non-infectious, whereas phages carrying in-frame inserts remain fully infectious and can hence be enriched by infection. This strategy was applied efficiently at a genome scale to generate an ORF-enriched whole genome fragment library from Mycobacterium tuberculosis, in which nearly 100% of the clones carried in-frame inserts after selection. The ORF-enriched libraries were successfully used for identification of linear and conformational epitopes for monoclonal antibodies specific to mycobacterial proteins

Alignment of the fragments of MTBLIB27 library selected by the monoclonal antibodies (A) Ag85-12 and (B) 1912.

<p>The translated regions of Ag85A, Ag85B and 19-kDa antigens of <i>M. tuberculosis</i> are represented as solid bar. The location of the deduced peptide sequences displayed on affinity-selected phages along with the position of the coded peptide is shown in bold on the right of the corresponding clone bar. The minimal overlapping sequence representing the putative epitope is shown below the alignment of each group.</p

Schematic representation of gIIIP protein encoded by the helper phages AGM13 and VCSM13.

<p>The N1, N2 and CT domains of the coat protein gIIIP are depicted as oval boxes (green) and are joined by the linkers L1 and L2. (A) A four-amino acid ‘KDIR’ trypsin cleavage site was introduced into both L1 and L2 linkers in the AGM13 helper phage, to encode trypsin-sensitive gIIIP. (B) VCSM13 is the wild-type helper phage without any trypsin cleavage sites, which encodes a trypsin-resistant gIIIP. The ‘KDIR’ amino acid sequence was inserted after residue 70 of gIIIP in L1 (shown in bold in A) and replaced four amino acids between residues 239–242 of gIIIP in L2 (shown in bold in B).</p

Distribution of <i>M. tuberculosis H37Rv</i> gene fragments in the MTBLIB27 library.

<p><b>5A</b>. Bar graph depicting the size distribution of <i>M. tuberculosis H37Rv</i> gene fragments in the MTBLIB27 at different stages of library construction. <b>5B</b>. Schematic representation of the distribution of gene fragments. The <i>M. tuberculosis</i> genome is ∼4.4 Mb and consists of ∼4000 genes. (I) MTBLIB27C01 primary cells before ORF selection; (II) MTBLIB27P01 after trypsin treatment (10 µg/ml) of primary phages; (III) MTBLIB27C02 secondary cells obtained after trypsin treatment (10 µg/ml) of primary phages for ORF selection; (IV) MTBLIB27P02 obtained from rescue of C02. The non-ORF selected inserts which align with the <i>M. tuberculosis</i> genome are shown as blue arrows; the clones in-frame with <i>PelBss</i> and <i>gIIIP</i> are indicated as red arrows (non-genic clones); the clones aligning with the <i>M. tuberculosis</i> proteome are indicated as green arrows (genic clones). The direction of the arrows indicates gene orientation. The maps are to scale.</p

Titre and reactivity of phages rescued by AGM13 and VCSM13.

<p>(A) The phagemid clones rescued with AGM13 and VCSM13 were titrated on <i>E. coli</i> TG1 and scored as the number of Amp^r transductants (CFU/ml); 3301 scFv is an antibody fragment specific to the protein MPT64 encoded by the <i>M. tuberculosis</i> gene <i>Rv1980c</i>; 19 kDa antigen is encoded by the <i>M. tuberculosis</i> gene <i>Rv3763</i>; and MAb 1905 is a monoclonal antibody against the 19 kDa antigen. (B) Different dilutions of rescued phages were added to Maxisorb™ plates coated with MPT64 (for 3301 scFv-displaying phages) or MAb 1905 (for 19 kDa antigen-displaying phages). The bound phages were detected using a HRP conjugated anti-phage MAb. Values are mean ± SD of three independent experiments. For Fig. 3B error bars are too small to be visible as compared to the physical size of the symbol.</p

Process of open reading frame (ORF) selection using the AGM13 helper phage.

<p>The gene-fragment library is constructed in a phagemid vector by inserting the fragments between the <i>PelB</i> signal sequence and full length native <i>gIIIP</i>, and transformants (C01, primary unselected library) are obtained in <i>E. coli</i> TOP10F’. (i) The C01 cells are used for producing phages using AGM13, which has a trypsin cleavage site (T) in the linker regions between the N1 and N2, and N2 and CT domains of gIIIP. During rescue, three types of phages are produced: A, Phages displaying protein matching with the <i>M. tuberculosis</i> proteome (genic clones) expressed as fusion protein with full length trypsin-resistant gIIIP; B, phages displaying protein not matching with the <i>M. tuberculosis</i> proteome (non-genic clone) but expressed as fusion protein with full length trypsin-resistant gIIIP. C, phages not displaying any protein as the insert is out-of-frame with the signal sequence and trypsin-resistant <i>gIIIP</i>, which carry only AGM13-encoded trypsin-sensitive gIIIP. (ii) Trypsin treatment of the phage population renders non-displaying phages (type C) non-infectious, while A and B become fully infectious due to removal of the displayed protein by cleavage of the trypsin site present between gIIIP and the fusion partner. Subsequent infection of the trypsin-treated library produces ORF-selected transductants (C02). (iii) Upon rescue with AGM13 or any other helper phage, the C02 transductants produce ORF-selected phages displaying protein, which could be from genic or non-genic DNA sequences.</p

Panning of whole genome fragment libraries on monoclonal antibodies raised against mycobacterial proteins.

<p>Control and Test refer to wells coated with phosphate buffer saline (pH 7.2) and specific monoclonal antibody, respectively.</p>*<p>Fold enrichment = output phage in test/output phage in control.</p>†<p>The inserts in clones from test wells selected after panning were sequenced and aligned to the respective <i>M. tuberculosis</i> protein.</p>#<p>All the clones analyzed were in frame with resepect to PelBss and gIIIP.</p

Analysis of random clones at various stages of library construction.

<p>Randomly selected clones were analyzed from (A) MTBLIB25 library (100–300 bp) and (B) MTBLIB27 library (300–800 bp) at various stages of library construction: 1, transformants obtained after large-scale electroporation of the ligation sample; 2, transductants obtained after infection of TOP10F’ cells with the primary phage library; 3, transductants obtained after infection of TOP10F’ cells with the trypsin-treated primary phages; 4, transductants obtained after infection of TOP10F’ cells with the secondary ORF-selected phage library.</p>*<p>Percentage of recombinant clones that aligned to the <i>M. tuberculosis</i> (<i>M. tb</i>) genome.</p>†<p>Percentage of clones in-frame with the PelB signal sequence (<i>PelBss</i>) and <i>gIIIP</i> in the phagemid.</p>‡<p>Percentage of total in-frame clones (as in III) that aligned with the <i>M. tb</i> proteome (genic ORFs).</p><p>Number of positive clones/total clones analyzed is given in brackets.</p