Synechocystis sp. PCC 6803 is the most popular cyanobacterial model for prokaryotic photosynthesis and for metabolic engineering to produce biofuels. Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insights into their metabolic potentials and regulatory networks. To enable a comparative approach, we generated the draft genome sequence of Synechocystis sp. PCC 6714, a closely related strain of 6803 (16S rDNA identity 99.4%) that also is amenable to genetic manipulation. Both strains share 2838 protein-coding genes, leaving 845 unique genes in Synechocystis sp. PCC 6803 and 895 genes in Synechocystis sp. PCC 6714. The genetic differences include a prophage in the genome of strain 6714, a different composition of the pool of transposable elements, and a ∼40 kb genomic island encoding several glycosyltransferases and transport proteins. We verified several physiological differences that were predicted on the basis of the respective genome sequence. Strain 6714 exhibited a lower tolerance to Zn2+ ions, associated with the lack of a corresponding export system and a lowered potential of salt acclimation due to the absence of a transport system for the re-uptake of the compatible solute glucosylglycerol. These new data will support the detailed comparative analyses of this important cyanobacterial group than has been possible thus far. Genome information for Synechocystis sp. PCC 6714 has been deposited in Genbank (accession no AMZV01000000). PCC 6803 (from here on Synechocystis 6803), the otherwise most popular cyanobacterial system to work with. Synechocystis 6803 was the first phototrophic and the third organism overall for which a complete genome sequence was determined.4 The genome of Synechocystis 6803 was manually curated by the research community at CyanoBase (http://genome.microbedb.jp/cyanobase/Synechocystis).5 Over the years, several substrains of 6803 evolved in different laboratories showing distinct physiological features (e.g. glucose tolerance), from which also several have recently been re-sequenced.9Genomic and transcriptomic comparisons between closely related bacteria are powerful approaches to infer insight into the metabolic potentials and regulatory networks. Among cyanobacteria, this has been illustrated by detailed comparative analyses of the marine picoplanktonic cyanobacteria 10 raising the number of publicly available cyanobacterial genome sequences to 126. With strain PCC 7509 also, one Synechocystis strain was sequenced. However, it is only very remotely related (90% 16S rRNA identity) to Synechocystis 6803 and belongs even to another clade (B1) than Synechocystis 6803 (B2) in the cyanobacterial tree.10 Therefore, despite its naming as Synechocystis, the strain PCC 7509 is quite distant from Synechocystis 6803. In the current cyanobacterial tree, Synechocystis 6803 is sharing a clade with unicellular N2-fixing oceanic strains such as Cyanothece spp.10 It has been reported that a 97–100% 16S rRNA identity is necessary for a productive genome comparison among strains.3The coverage with analysed genome sequences for the cyanobacterial phylum has been greatly improved recently. Based on a diversity-driven selection of species for genome sequencing, 54 additional strains were analysed,Synechocystis 6803 lacked a closely related organism with a known genome sequence that appeared suitable for comparative analysis. To fill this gap, we selected Synechocystis sp. PCC 6714 (from here: Synechocystis 6714) as candidate. Synechocystis 6803 as well as strain 6714 are unicellular cyanobacteria that were isolated from the same freshwater pond in Oakland, California, by R. Kunisawa. These strains were initially part of the ‘Berkeley Culture Collection’,11 which were later transferred into the ‘Pasteur Culture Collection’ of cyanobacteria.12 The decision to choose Synechocystis 6714 was further supported by the high 16S rRNA identity (99.4%) among the two strains, thus well suited for comparative analyses. Their close genetic relation also was seen in an expression-based screen that revealed the presence of a highly transcribed CRISPR system in it,13 similar to the one in Synechocystis 6803.14 Moreover, the strain 6714 also represents an established laboratory strain, amenable to genetic manipulation.16Thus, Synechocystis 6714 in comparison to strain 6803. In a parallel study, we will provide the primary transcriptomes of both strains under 10 different conditions using strand-specific cDNA sequencing.Here, we focus on the draft genome analysis of 2.2.1.Synechocystis 6714 was purchased from the Pasteur Culture Collection (PCC) in Paris, France. Genomic DNA was extracted as described earlier.9 We prepared two libraries for sequencing, one with fragment lengths of 160 nt for paired-end sequencing and one with ∼3 kb long fragments, which was used for preparing a mate pair library . Both libraries were subjected to paired-end sequencing, yielding 135 969 158 reads of 101 nt length. The accumulated sequence information resulted in a nearly 2000-fold coverage when expecting a genome of 3.5 Mb. The reads were assembled with velvet17 using a kmer-length of 85, a coverage cut-off at 5, and an expected coverage of 1300. This resulted in 74 contigs arranged in five scaffolds longer than 10 000 nt. Gaps within scaffolds were analysed by polymerase chain reaction (PCR) and subsequent Sanger sequencing and, if successful, closed with the obtained sequence information. This removed 19 gaps reducing the number to 55. Gene prediction and annotation was done with RAST18 and led to the prediction of 3733 protein coding genes, 40 tRNAs, and 1 cluster of ribosomal RNAs.2.2.Synechocystis 6714 and 3683 ORFs from Synechocystis 6803, resulting from a combination of all ORFs from Mitschke et al., Supplementary data file S119 and all plasmid-located ORFs annotated in CyanoBase5 . Orthologues of protein coding genes were identified using a reciprocal best blast hit (RBH) strategy. For the identification of gene families and unique genes, we used Markov clustering (MCL)20 on the results of reciprocal BlastP searches. Clustering into protein families using the MCL algorithm yielded 2413 shared protein families with 3385 and 3187 members in Synechocystis 6803 and 6714, respectively. Putative transposase genes were identified by BLASTp searches against the ISfinder21 requiring a BLASTp value of ≤1e10−8.Orthologue prediction was based on the 3733 open reading frames (ORFs) from 2.3.Synechocystis 6714, we used Synechocystis 6803 substrain ‘PCC-M’9 for comparative physiological experiments. Liquid cultures were grown at 30°C in liquid BG11 medium12 under continuous white light illumination of 50–80 µmol quanta m−2 s−1. For growth on solid medium, BG11 was supplemented with 0.9% agar . Salt-dependent growth, GG contents, and mRNA patterns were measured for cultures of 300 ml volume that were grown under constant shaking in 500 ml Erlenmeyer flasks. The cells were pre-cultivated for 2 days at standard conditions before sterile, crystalline NaCl was added to a final concentration of 2, 4, and 6% (w/v), respectively. After 4 days, 50 ml of cells was harvested by rapid filtration on hydrophilic polyethersulfone filters . The adherent cell material was immediately dissolved in 1 ml of PGTX solution22 and total RNA was extracted as described.13 For the measurement of glucosylglycerol content, 2 ml of cells was harvested by centrifugation and soluble metabolites were extracted with 80% ethanol . The supernatant was freeze-dried. The cell extract was then purified from insoluble material by centrifugation and the supernatant was also freeze-dried. Both, the dried cell extracts and external fractions were resuspended in A. dest , centrifuged and the supernatant was freeze-dried again. Samples were then analysed by gas chromatography as previously described.23In addition to 2.4.32P-labelled RNA probes generated from specific DNA templates by using Ambion® MAXIscript® T7 In Vitro Transcription Kit as described earlier.24 The oligonucleotide sequences used for the generation of DNA templates by PCR are given in Supplementary Table S1. Signals were visualized with the Personal Molecular Imager FX system and Quantity One software (Bio-Rad).For expression analysis, 3 µg of total RNA was separated on 1.5% agarose gels, transferred to Hybond-N nylon membranes by capillary blotting and cross-linked by UV-illumination. The membranes were hybridized with 3.3.1.Synechocystis 6714 was sequenced using two libraries of different lengths by paired-end sequencing and assembled into five scaffolds ranging from 46 504 to 2 984 476 nt in length. The DNA is characterized by an average GC content of 47.37%, which is very close to the value of 47.4% reported for strain 6714 in 1971 based on CsCl density gradient equilibrium centrifugation.11 The longest scaffold C2 likely represents the major part of the chromosome, since it closely resembles the chromosome of strain 6803. Table Synechocystis 6803, they also are likely part of the chromosome. This assumption also is in line with their 3 and 5% higher GC content compared with the scaffolds C1 and C3. For comparison, the GC content of the Synechocystis 6803 chromosome is 47.72%, whereas it also is lower for three out of the four large plasmids .25 By combining the scaffolds C0, C2, and C4, we estimated the size of the Synechocystis 6714 chromosome to be around 3.45 Mb, which is fairly similar to the 3.57 Mb of Synechocystis 6803.The genome of 28 of Synechocystis 6803, we found no significant similarities toward its plasmids pSYSG, pCC5.2, pCA2.4, and pCB2.4 in strain 6714. In contrast, sequences resembling about one-third each of pSYSA, pSYSM, and pSYSX of Synechocystis 6803 were detected in strain 6714 . In Synechocystis 6714, we identified only 32 transposase genes, which belong to only six different families. The highest copy numbers were found for the IS200/IS605 family and as before in strain 6803 for IS630 and IS5 families (Table Supplementary Table S3). At a first glance, this high divergence in the numbers and types of insertion sequences appears surprising, given the otherwise close relatedness among the two strains. However, this finding is in line with reports for the ISY203 group of elements (belonging to the IS4 family) that vary even among substrains of 6803. Four members of this IS element with identical nucleotide sequences were present only in the ‘Kazusa’ substrain, whereas they were absent in the genomes of other substrains.29A marked difference between both strains exists in the number and types of mobile genetic elements Table . SynechoSynechocystis 6714 and 6803, leaving 845 specific genes in strain 6803 and 895 specific genes in strain 6714. Thus, among the two strains, more than 75% of the genes are conserved. However, many of the strain-specific genes belong to gene families that were clustered as paralogues to pairs of orthologue genes when using the MCL algorithm,20 indicating gene duplications, sequence, and probably also functional diversification. The full list of orthologue and paralogue genes between the two strains is presented in Supplementary Table S4. In Supplementary Tables S5 and S6, we present the lists of unique protein-coding genes. Only 537 of the 845 Synechocystis-6803-specific genes are located on the chromosome , whereas 308 of the genes lacking a clear orthologue  can be explained by the strong differences in the plasmid-located gene pool. Moreover, it should be noted that the majority of strain-specific genes encodes for proteins of unknown function, i.e. the functional significance of the majority of differences is thus uncertain.Using RBH, we identified 2838 orthologous protein-coding genes between 3.2.Synechocystis 6803 is correlated with a low degree of syntheny between the two strains. Another situation exists with the rfb-gene cluster that differs entirely between the two strains and encodes several glycosyltransferases possibly involved in cell wall biosynthesis and the modification of cell surface properties. This region has features of a genomic island, since the adjacent genes are conserved between the two Synechocystis strains, but the GC content drops considerably (from 48 to 35%) within this region in both strains  and several Thioalkalivibrio species, pointing further to the alien origin of this genomic region.The higher number of transposon genes in ins Fig. . Genomicsll1220–sll1226  in strain 6803. In Synechocystis 6714, the orthologues of these seven genes (sly1009900–sly1009960) form a cluster with gene sly1009870 encoding the NiFe hydrogenase metallocenter assembly protein HypD, whereas the homologue in Synechocystis 6803, slr1498, is located 1.62 Mb away.An example for genome scrambling worth mentioning exists in the hydrogenase operon that encompasses the seven genes Synechocystis 6714 and 6803 genomes is the presence of a prophage in the former but its lack in the latter  gene, duplicating its 3′ half but restoring the gene to be functional intact. This insertion might have occurred only recently as the duplicated segment of the trnF gene is still sequence identical with the original prophage host gene. Although the Psy1 genome is with a total length of 20 660 nt quite short for a prophage, genomes of comparable size have recently been reported for siphoviruses, which infect marine cyanobacteria .31 The annotation of Psy1 adds another 27 genes unique for strain 6714 (Supplementary Table S6). Most of these genes have no closely related homologues in database searches, indicating that Psy1 might belong to a novel group of bacteriophages. Clear homologues exist for Sly1027750, an integrase with several homologues in other cyanobacterial genomes; Sly1027640, an HK97 family phage portal protein with the tail sheath protein from the Pseudomonas transducing phage PhiPA3 as the best matching protein in the bacteriophage database ;32 Sly1027490, a lysozyme superfamily protein with the putative endolysin from Acinetobacter phage phiAC-1 as the best matching bacteriophage protein ;33 Sly1027700, a D5 N terminal like domain-containing protein of phage D5 proteins and bacteriophage P4 DNA primases . Interestingly, gene ssl7074 in Synechocystis 6803 is located within the CRISPR2-associated region of cas genes next to the cas 6-2b gene, a candidate for an endonuclease involved in CRISPR crRNA maturation.14Our complementary transcriptome data (unpublished) indicate that the Psy1 genes are not significantly expressed except for a short region, encompassing the two short genes 680 Fig. . One of 3.3.Supplementary Table S5 and S6 for the list of unique genes in Synechocystis 6803 and 6714, respectively). For instance, only Synechocystis 6803 possesses genes for the proteins Flv2 and Flv4, which are essential for growth under fluctuating light and are supposed to protect photosystem II against photoinhibition.34 In contrast, in Synechocystis 6714, two operons are found, each encoding all subunits of the high-affinity K+ transporter Kdp,35 similar to the situation in filamentous cyanobacteria such as Anabaena sp. PCC7120, whereas Synechocystis 6803 harbours only one copy of the kdp2 type.36 To date, it was believed that unicellular cyanobacteria have a single kdp system or none, whereas filamentous cyanobacteria have two or more copies.36Several of the strain-specific genes likely affect the physiology or provide certain strain-specific characteristics allowing their settlement in specific environmental niches (see Supplementary Tables S5 and S6). There are three distinct loci of CRISPR-cas genes in both strains.14 One of them  is highly conserved, whereas the other two appear to have been substituted over their entire length, possibly by an active mechanism of exchange. Details of the different CRISPR-cas loci were published separately.13A distinct group of protein-coding genes that differs between the two strains are associated with the CRISPR system, the prokaryotic immune system, accounting for 17 different proteins alone , a type 4 pilin-like protein, which is involved in the formation of thick pili and motility40 and therefore may explain the observed phenotype.One feature that has been reported to differ even between 2+ and Co2+ 42 appears to be specific for Synechocystis 6803 and missing in strain 6714. The functional significance of this difference was tested in growth experiments in the presence of increasing amounts of Zn2+ ions and revealed the higher tolerance of strain 6803 against high Zn2+ levels uptake of the compatible solute glucosylglycerol.44 Apart from that, the loci adjacent to ggtA or ggtBCD in Synechocystis 6803 are conserved in the genome of Synechocystis 6714 , the cells of both strains were virtually free of GG. In Synechocystis 6803, the intracellular GG level increased corresponding to the external salt level, whereas in the surrounding medium, virtually no GG was found  were similar. However, no further increase was observed when cells were grown at 6% NaCl pointing to a somewhat lower salt tolerance of strain 6714 (see below). Interestingly, GG also accumulated in high amounts in the surrounding medium, which supports the assumption that an effective system for the re-uptake of GG is missing in Synechocystis 6714 46 are found in the genome of Synechocystis 6714. However, a homologue of sll1685 (PxcA) which might be involved in the energetization of Na+ transport is missing. Furthermore, the genome of Synechocystis 6714 harbours two copies of the kdp operon each encoding a high-affinity K+ transporter (genes sly5000010–sly5000050 and sly1021590– sly1021630), whereas strain 6803 has a single copy of this operon (slr1728–slr1731). The Kdp ATPase system, initially characterized in Escherichia coli, is responsible for the immediate uptake of K+ after salt or osmotic shock in E. coli.35 In combination with glutamate as an organic counter ion, K+ is believed to act as a temporary compatible solute and moreover as a regulatory signal for the initiation of subsequent acclimation processes, also in cyanobacteria.47 Interestingly, the kinetics for the uptake of K+ in cyanobacteria after salt shock was characterized for Synechocystis 6714.48 A sudden osmotic shift by adding 500 mM NaCl was followed by a transient accumulation of K+ which started within the first minutes, peaked at around 30–60 min and declined after 24 h to levels similar to non-shocked cells. The decrease in K+ was accompanied by an accumulation of GG. The kinetics of a K+ uptake have not been measured so far for Synechocystis 6803, but it might be a bit different from the process in Synechocystis 6714 due to the absence of a second kdp operon.In addition to compatible solute accumulation, a balancing of the ionic composition is also important to cope with changing salinities. For instance, an active extrusion of Na50 Interestingly, glycosyltransferase and glycoside hydrolase gene families have also been found frequent in several of these cyanobacterial genomic islands. Therefore, the modification of cell surface polysaccharide and lipopolysaccharide biosynthesis by several of these enzymes, presumably allowing diversification of cell surface features appears central for this group of organisms. Such modification capacity is likely to be relevant in the avoidance of grazers and even more in the avoidance of bacteriophage infection.51Another interesting observation is the putative substitution of a gene cassette of ∼40 kb encoding several glycosyltransferases, transport proteins, and hypothetical proteins in the two strains. Together with the presence of some genes not found in any other cyanobacteria and the strongly reduced average GC % content in this region, this region is likely representing a genomic island. Physiologically and ecologically important genomic islands have been identified in several marine cyanobacteria.Synechocystis 6714 allows to follow interesting research problems in this strain. However, most importantly, it opens exciting new opportunities when working with the most advanced cyanobacterial model, Synechocystis 6803.In conclusion, the draft genome analysis of 5.Synechocystis sp. PCC6714 genome are available under the accession no. AMZV01000000 at Genbank. The annotated version including also short assembled regions is available at http://www.cyanolab.de/Supplementary.html.The assembled scaffolds of the Supplementary Data are available at www.dnaresearch.oxfordjournals.org.the Federal Ministry of Education and Research grant ‘e:bio RNAsys’0316165 (to W.R.H.).The research leading to these results has received funding from


Despite impressive decreases in under-five mortality, progress in reducing maternal and neonatal mortality in Tanzania has been slow. We present an evaluation of a cadre of maternal, newborn, and child health community health worker (MNCH CHW) focused on preventive and promotive services during the antenatal and postpartum periods in Morogoro Region, Tanzania. Study findings review the effect of several critical design elements on knowledge, time allocation, service delivery, satisfaction, and motivation.n = 88) and (2) CHWs (n = 53) identified in the same districts prior to the program’s start. Service delivery outputs were measured by register data and through a time motion study conducted among a sub-sample of 33 randomly selected MNCH CHWs.A quantitative survey on service delivery and knowledge was administered to 228 (of 238 trained) MNCH CHWs. Results are compared against surveys administered to (1) providers in nine health centers (n = 228) were interviewed: 55% male, 58% married, and 52% with secondary school education or higher. MNCH CHWs when compared to earlier CHWs were more likely to be unmarried, younger, and more educated. Mean MNCH CHW knowledge scores were <50% for 8 of 10 MNCH domains assessed and comparable to those observed for health center providers but lower than those for earlier CHWs. MNCH CHWs reported covering a mean of 186 households and were observed to provide MNCH services for 5 h weekly. Attendance of monthly facility-based supervision meetings was nearly universal and focused largely on registers, yet data quality assessments highlighted inconsistencies. Despite program plans to provide financial incentives and bicycles for transport, only 56% of CHWs had received financial incentives and none received bicycles.Ninety-seven percent of MNCH CHWs  contains supplementary material, which is available to authorized users. Child mortality rates in Tanzania have declined by nearly 70% over the last 25 years and in 2014 nearly reached the Millennium Development Goal (MDG) 4 target of 55 per 1000 live births .Key contributors to the slow progress in reducing neonatal and maternal mortality have been (1) stagnant levels of facility deliveries, 2) poor quality of care, (3) lack of contact of health services with children during their first 28 days of life, and 4) breaks in the continuity of care from preconception through antenatal, intra-partum, and postpartum periods. Critical shortages of health workers underpin these contributing factors, impeding efforts to improve timely and continuous access to high-quality health services in Tanzania and many other low-resource settings. When compared to the WHO-recommended health workforce density of 25 health professionals  per 10 000 people, Tanzania lags behind with only 4 health professionals for every 10 000 citizens [ poor qua breaks iujamaa (collectivized) villages, which in part aimed to make social services more accessible to rural populations [The use of CHWs in Tanzania began in the 1960s alongside President Nyerere’s implementation of ulations . Early pulations . Howeverulations . In 1983ulations . These pSince the 1980s, CHW programs in Tanzania have concentrated heavily on narrow mandates determined by funders, often focusing on only one health problem, most commonly HIV/AIDS ,7. WhileIn 2012, the MoHSW approved national guidelines for training maternal, newborn, and child health (MNCH) CHWs as part of an interim solution until a single national cadre of CHWs could be established. These volunteer MNCH CHWs are selected by the community, trained for 21 days, and supervised by facility providers to deliver a range of preventive and promotive services during the antenatal and postpartum periods through home visits and community meetings. This socially orientated CHW is in contrast to the more medically orientated integrated community case management (iCCM) CHW models being established in a number of other countries in the region .These CHW model variations require different provider competencies. For iCCM CHWs to be effective, CHWs need to be able to correctly identify, assess, and treat sick children based on national guidelines, and systems need to be in place to ensure adequate clinical supervision and medical supplies. In contrast, for a preventive-promotive MNCH CHW to be effective, it is necessary to master a large amount of information, understand which messages are appropriate for visits at different points in the continuum of care, and reach a high proportion of women and children at each point in the continuum. For more comprehensive CHW models, which draw upon a range of both medically and socially orientated tasks, CHWs must achieve competency in all of these tasks.This paper profiles a recently established cadre of socially oriented MNCH CHWs that provides health preventive and promotive MNCH services in Morogoro Region, Tanzania. We review several critical design elements including CHW profile and MNCH knowledge, CHW to population coverage ratio, and program monitoring and supervision and assess their implications on time allocation, service delivery, satisfaction and motivation, and incentive preferences.2, making it the sixth most populous and second largest of the country’s 25 mainland regions [Two hundred kilometers west of Dar es Salaam, Morogoro Region is home to over 2.2 million people dispersed over 70 000 km regions . Seventy regions . Over ha regions . In the  regions .Implemented by the MoHSW with support from Jhpiego and established through the USAID-funded Mothers and Infants, Safe, Healthy and Alive (MAISHA) program, the Integrated Community to Facility MNCH Program aims to improve access to and quality of maternal, newborn, and reproductive health services. Integrated MNCH CHW Program training activities began in 2010 with a 6-day training of health center providers (mean of 2–4 per facility) according to facility-based guidelines  MNCH CHWs following their recruitment, training, and deployment (Table 8 MNCH CHn = 88) and (2) CHWs identified in the same districts prior to the program’s start at the community level (n = 53). These comparisons were intended to spur discourse on MNCH CHW eligibility criteria and provide broader insights into MNCH CHW competency and service delivery. RCH providers in nine health centers (n = 88) were interviewed during a facility assessment survey conducted in 2012. In 2011, prior to the rollout of MNCH CHWs, a CHW census was carried out to determine the number of providers and assess knowledge and service delivery of individuals who self reported and/or were said to be CHWs (n = 53) by key stakeholders including village leaders and facility-based providers. Once identified, research assistants administered a quantitative survey exploring personal characteristics, working conditions, incentives, knowledge, motivation, and job satisfaction.MNCH CHW knowledge results were compared against knowledge surveys administered to two populations of providers operating in the same geographic area: (1) health center reproductive child health (RCH) providers (n = 33) of MNCH CHWs randomly selected from among those interviewed for the quantitative survey. Observations sought to improve understanding of the frequency and content of MNCH service provision, including use of job aidsa, as well as the broader context within which services are provided. The time motion study was constrained to the CHW’s village of residence and spanned for a period of up to six consecutive days beginning on a Wednesday and ending on a Monday in most instances. During the period of observation, a team of independent research assistants observed and continuously timed all activities carried out between the hours of 8 am and 5 pm. Activities performed outside of the observation window (from 5 pm to 8 am) were self-quantified at the start of each new day and recorded as “reported time allocation.” Findings are presented only on observed time allocation.Service delivery outputs were measured by extracting data from the Health and Management Information System (HMIS) registers of interviewed MNCH CHWs for the 5 months preceding the survey (May to September 2013) and through direct observations. For the latter, a time motion study was conducted from December 2013 to January 2014 among a sub-sample of ~15% (n = 33), we compared MNCH monthly summary sheets for 3 months with the maternal and child health (MCH) registers for the same 3 months for each of the 33 CHWs. Summary sheets form the basis of reported service delivery statistics and are submitted by individual MNCH CHWs to supervisors monthly and ultimately aggregated across all MNCH CHWs. The MNCH CHW summary sheets were assessed for discrepancies (over or under reporting) with the MCH registers in the number of the following visits: new pregnant women, returning pregnant women, neonates, children 1–12 months old, children 12–59 months old, and total home visits. For each type of visit, we calculated the number of CHWs with discrepancies and the magnitude of these discrepancies. We also assessed for patterns of discrepancies by CHW gender, education, and date of training.To assess the quality of HMIS registers, among the MNCH CHWs observed during the time motion study . Ordered logistic regression models were used to explore associations between MNCH CHW characteristics  and composite knowledge scores overall and across domains. An asset index was constructed from CHW household assets and characteristics, using principal components analysis. Time motion data were analyzed using basic frequencies and cross tabulations.The study received ethical approval from the Muhimbili University of Health and Allied Sciences and Johns Hopkins School of Public Health Institutional Review Boards. Preliminary findings were shared with key decision makers in Tanzania from the MoHSW and Jhpiego for their feedback and review prior to publications being drafted.n = 228) reported as trained between December 2012 and July 2013 were identified and successfully interviewed based on personnel lists provided by the Integrated MNCH CHW Program. Fifty-five percent of MNCH CHWs were male, all were Swahili speakers (100%), 58% were married, and 52% had secondary school education or higher (Table Ninety-seven percent of the CHWs n = 28 reportn = 53), prior to the start of MNCH CHW program, the ratio of male to female CHWs was similar over time. However, differences in age, education, and marital status were observed. In 2011, CHWs were more likely to be married (75% in 2011 versus 58% in 2013), were older (mean age of 41 in 2011 as compared to 32 in 2013), and were less educated (17% had secondary school education or higher in 2011 as compared to 52% in 2013).When compared against CHWs interviewed in 2011 , while postpartum care (40%), HIV transmission (37%), and nutrition (35%) were the lowest  and MNCH CHWs (50%) but lower than those for CHWs interviewed in 2011 (64%) . Assuming a 20-day work month, CHWs would need to conduct almost two household visits per day. In the event of an 8-day work month, CHWs would be required to conduct nearly five household visits per day.CHWs reported having a mean of 186  households per catchment area to cover, corresponding to approximately 1 CHW per 967 people (median 1 per 624 population). Assuming a birth rate of 30 per 1000, this corresponds to an estimated 29Systems for tracking MNCH CHW service delivery were established through the introduction of MNCH CHW HMIS registers: (1) maternal and child health (MCH) and (2) referral register. Key summary indicators for the former are outlined in Table n = 228) was currently recording information into the MCH register, and 98% was recording information in the referral register. For the MNCH CHWs observed during the time motion study (n = 33), summary sheets for a 3-month period generated by trained research assistants were compared against those recorded by MNCH CHWs. The quality of data was found to be variable across the eight indicators (type of visits) assessed , which is approximately between 2–3 households per day during a median 2 days of work per week Table . Of the P < 0.8). However, trends in the mean number of households visited per month per CHW were observed to decline slightly over time with the concurrent growth in the number of MNCH CHWs providing services.CHWs reported having at least one other MNCH CHW and an average of 3.7 other CHWs working in the same area. A significant association between mean households served and mean monthly home visits reported was not observed  reported attending monthly supervision meetings held in health centers or dispensaries continuously since receipt of training. High rates of attendance may in part be attributed to financial incentives disbursed during these monthly meetings  and quarterly supervision visits ) which total 50% of the MNCH CHWs’ median monthly household income .All interviewed CHWs (n = 46) trained in December 2012/January 2013, a mean of 1.5 of the three scheduled quarterly visits had taken place. Nearly all CHWs trained (n = 86) in April/May 2013 had received their scheduled quarterly supervision visit (CHWs reported a mean of 0.76 quarterly visits).Facility-based providers also were reported to visit CHWs in the village a mean of once in 2 months. Planned quarterly supervision from Jhpiego/MoHSW regional and district providers occurred with less frequency. Among CHWs . Knowledge assessments, feedback on work performance, work planning, and/or additional training were mentioned with less frequency (<50%).Thirty-three MNCH CHWs were observed: 29 for a total of 6 days and the remaining for less than 5 days. MNCH CHWs were directly observed to spend 6.75 (15%) out of 43 h on health service delivery, 11% (5 h) on MNCH services, and 4% (1.75 h) on other health work. Given the financial incentives paid by the MNCH CHW program, time spent on the MNCH CHW program corresponds to 1,750 TSH or US$ 1.06 per hour and compares favorably to the estimated hourly wage of 1,805 TSH or US$ 1.09 earned through time spent on alternative income-generating activities.Of the 5 h CHWs spent on MNCH activities, 70% was indirectly related to client care  and 30% on home visit consultations. CHWs were observed to provide home visits to a mean of two clients per week with each visit ranging from 75 min for a pregnancy visit, 86 min for a postpartum visit, and 19 min for a follow-up visit made 5 months after the immediate postpartum period.During 37 observed pregnancy visits, 26 different job aides were used, 3 with ~80% frequency: individual birth preparedness (IBP), pregnancy danger signs, and maternal nutrition. Two job aids recommended for use during all pregnancy visits—pregnancy danger signs and prevention of mother to child transmission—were missed in 19% and 38% of home visits, respectively. Thirty-three percent of job aids observed were not those recommended for pregnancy visits but instead cited as optional content. During nine observed postpartum visits, 14 different job aides were observed, 6 with nearly 80% frequency: malaria prevention, lactational amenorrhea method, nutrition, child danger signs, accident prevention, and infection prevention. The single job aid on breastfeeding recommended for use in all postpartum home visits was missed in 23% of visits.a, and 80% of CHWs were dissatisfied with financial incentives provided [Almost all (99%) CHWs interviewed reported being happy to work as CHWs and over 90% felt their work to be valued by both the health facility workers and the community. High levels of satisfaction were reported for the availability of job aids (90%) and registers (91%), level and quality of training received (90%), and quality of their own work (88%) . Almost provided .MNCH CHWs were intended to receive 25,000 TSH (US$ 15.00) for attending monthly meetings and 10,000 TSH (US$ 6.00) for attendance of quarterly visits from Jhpiego and regional and district MoHSW staff. When provided, this amount corresponds to an estimated 50% of the median monthly household income of 50,000 TSH (US$ 28.78) reported by CHWs. When asked about incentives received, slightly more than half (56%) of CHWs had received financial incentives since their training, but none had received the bicycles promised to facilitate transport. When asked their preference, non-monetary incentives are relatively more important than monetary incentives for CHWs. Specifically, community recognition and respect were most preferred over other incentives. However, CHW remuneration and retention remain a concern, as 60% of CHWs feel overburdened due to other competing household and professional responsibilities to the extent that 14% indicate that they contemplated quitting. Younger CHWs were more likely to feel overburdened and less valued by community members.Building upon five decades of CHW programs in Tanzania, the Integrated MNCH CHW Program sought to establish a volunteer cadre of MNCH CHWs that provide a range of socially oriented, preventive, and promotive services including village mapping, pregnancy surveillance, counseling through home visits, and health promotion meetings. This model is in contrast to other current CHW initiatives like the iCCM model, which expands on preventive and promotive activities to include curative services, requiring clinical oversight, training, and support inclusive of more sophisticated supply chain mechanisms. We examine how time allocation, service delivery, satisfaction, and motivation are effected by the following critical design elements (1) CHW profile and MNCH knowledge, (2) CHW to population ratio, (3) program monitoring and supervision, and (4) incentives.CHW characteristics, including age, gender, education, and martial status, may influence performance ,15. At iOverall mean knowledge scores for MNCH CHWs were observed to be poor at less than 50% for 8 of 10 MNCH domains assessed. However, set standards for interpreting knowledge scores for CHWs are not available within the literature to enable comparison between studies. Job aids may temper the effect of these gaps in CHW knowledge during counseling, and supportive supervision and fresher training help to overcome them. However, additional efforts are needed to prioritize key messages based on evidence and to assess whether changes in content or duration of training might lead to improvements in knowledge, ultimately translating to better quality of counseling. A 2010 review by WHO and the Global Health Workforce Alliance identified 19 studies which assessed CHW knowledge, attitudes, and practices . HoweverIn the absence of global standards for CHW knowledge and a wider array of examples in the literature , we usedBeyond the contextualization of knowledge scores against those observed for other providers, we note that our data do not allow us to link knowledge scores to outcome and impact level indicators. Further, findings from Morogoro Evaluation Project (MEP) facility assessment activities in 2012 suggest that provider knowledge may not translate to improvements in the content of services provided. Rather, they suggest greater complexities may influence service delivery extending above and beyond what one “knows,” including provider and client perceptions, client characteristics, and the availability of provider time for service delivery given the high patient volume and competing time demands, among other factors  . The pooStudies elsewhere suggest that CHW performance is higher when the CHW to population coverage ratio is lower . Wide vaP < 0.8), MNCH CHWs provided services for a mean of 2 days per week—effectively part time. For alternative, more intensive CHW models, setting the number of CHWs to a population ratio versus village number may serve to reduce variability in coverage, content, and quality of care.At the national level, as dialogue continues on how many CHWs to train per village or population, the implications on individual workload need to be considered given the wide variations in village sizes across Tanzania. While we did not observe a significant association between mean households served and mean monthly home visits reported  and at high coverage is needed before recommendations on the appropriateness of the MNCH CHW program for delivery at scale can be made. A more robust evaluation, which considers elements of the quality of CHW counseling, timing, and coverage of home visits along with changes in facility-based utilization, is recommended to inform decision-making on MNCH CHW implementation moving forward.This study profiles MNCH CHWs in Morogoro Region and provides evidence on key operational concerns for CHW programming. Further research is required to understand the balance of CHW performance, CHW to population ratio, and incentives before scale up at the national level. The social profile of CHWs was not significantly associated with CHW knowledge or service delivery suggesting that a broader range of community members could be considered as CHW candidates pending community acceptance and fulfillment of competency requirements. Efforts to increase the service delivery outputs of MNCH CHWs will need to counter incentives CHWs have to devote time to alternative, and often more lucrative, income-generating opportunities. Given prior research demonstrating the existing poor quality of pregnancy and postpartum care at the facility level, efforts to scale up CHW programs need to equally prioritize the strengthening of facility support and service delivery. Despite heavy emphasis on registers during supervision, quality of record keeping was poor and the performance indicators measured were not optimal for tracking the timing of service delivery. CHWs, while highly motivated, also had expectations raised by the program for transportation and other financial incentives, which were not met. These early experiences with MNCH CHW implementation have provided insights into possible areas for program improvement and lessons for scale up.aTo guide counseling during MNCH CHW home visits, CHWs are provided with 26 different job aides. During pregnancy, 17 different job aids are recommended for use during three different home visits (Web figure 3). In the postpartum period, 22 jobs aids are suggested for use at different time points during seven home visits recommended up to 5 months postpartum. Nine job aids are suggested for use during follow-up visits recommended from 5 to 59 months following delivery. While assessment of the quality of counseling was beyond the scope of observations, we observed which job aids were administered during home visits.bIf median estimates are used, this corresponds to an estimated 19 pregnancies and 17 newborns annually.


Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) has been reported to be a novel surgical technique that provides fast and effective growth of liver remnant. Despite occasional reports on animal studies, the mechanisms of rapid liver regeneration in ALPPS remains unclear. In the present study, we intend to develop a reproducible rat model to mimick ALPPS and to explore the underlying mechanisms. Rats assigned to the portal vein ligation (PVL), left lateral lobe (LLL) resection, transection and sham groups served as controls. Results indicated that the regeneration rate in the remnant liver after ALPPS was two times relative to PVL, whereas rats with transection alone showed minimal volume increase. The expression levels of Ki-67 and PCNA were about ten-fold higher after ALPPS compared with the transection and LLL resection groups, and four-fold higher compared with the PVL group. The levels of TNF-α, IL-6 and HGF in the regenerating liver remnant were about three-fold higher after ALPPS than the controls. There was a more significant activation of NF-κB p65, STAT3 and Yap after ALPPS, suggesting synergistic activation of the pathways by PVL and transection, which might play an important role in liver regeneration after ALPPS. Hepatic resection is still one of the most effective treatments for liver tumors35et al. developed a reproducible mouse model mimicking ALPPS and explored the underlying mechanisms. Their results suggested that ALPPS in rats induces an unprecedented degree of liver regeneration, comparable with humans. PVL in combination with the circulating factors seem to mediate the enhanced liver regeneration after ALPPS12The associating liver partition and portal vein ligation for staged hepatectomy (ALPPS) procedure is a new surgical strategy. It represents a modification of a two-stage hepatectomy in which complete parenchymal transection is associated with ligation of contralateral portal vein71112The rat liver is divided into the right lobe, median lobe, left lateral lobe, and caudal lobe . Each lo%. In the ALPPS group, it increased to 0.92 ± 0.11%  on day 3, reaching 1.81 ± 0.23%  on day 7  levels as established markers of hepatocyte injury were measured and the part of the occluded RML was examined using H&E staining. Histological studies after the PVL and ALPPS revealed areas of necrosis in the occluded part of the RML, which were significantly larger after ALPPS than after PVL on day 2  as shown in To investigate the mechanisms of liver regeneration after different surgical procedures, the expression changes of liver regeneration relative genes were investigated using ELISA . ResultsAs previously reported, studies of gene-expression cascades on the molecular mechanisms in the regenerating liver have provided insights into the signaling pathways that are rapidly activated in the liver remnant after hepatectomy or PVL151718A major advantage of ALPPS is the possibility for an early completion of Step II in clinical practice, that is, removing the deportalized liver within 2 weeks after Step I. In our study, seven animals which were allocated to the ALPPS group showed a normal liver remnant to bodyweight ratio (LR/BW ratio) 2 days after Step II or 9 days after Step I. This fast rate of liver regeneration was consistent with a previous reportThe size of the FLR is an important limiting factor for major liver resections201112et al. suggested that transection of the parenchyma may induce an inflammatory response with the release of putative growth factors, which may enhance the regenerative process18Although there are lots of ongoing debates regarding the mechanisms of the accelerated liver regeneration after ALPPS, the mechanism for this phenomenon is not clear and warrants detail mechanistic investigation. As reported, liver regeneration is an event that involves multiple cellular processes and has a complex interaction with cytokines and growth factors14in situ transection, the entire portal flow is redistributed towards the FLR, thus constituting a stimulus to regeneration; b) the local trauma caused by liver partition (transection) can also be a stimulus; c) a synergistic activation of the liver regeneration-associated signaling pathways, such as cytokines and transcription factors, contribute to the significant liver regeneration after ALPPS. At present, the mechanisms for rapid promotion of hepatic regeneration after ALPPS is not clear, and the procedure is still in its initial stage2526In summary, we speculated that there might be several possible explanations for the highest liver regeneration response in the ALPPS group: a) after complete separation of the LML by PVL and In this study, 480 male Sprague-Dawley rats weighing between 200 and 250 g were used. All the rats were provided with unlimited access to food and water before and after treatment. All surgeries were performed under anesthesia. The experimental protocol was reviewed and approved by the Committee on the Use of Live Animals in Teaching and Research of the Harbin Medical University, Harbin, China (SYSK 2010-012). We confirmed that all experiments were performed in accordance with relevant approved guidelines and regulations.In the current study, the rats were randomly divided into the sham group, the LLL resection group, the transection group, the PVL group, and the ALPPS group. The rats were fasted 12 h before the operations. All operations were performed under an operating microscope . After anesthesia, the abdomen was opened through a transverse incision. The liver was dissected from its ligaments. Briefly for the ALPPS procedure, dissection of the caudate lobe followed by ligation of the portal vein supplying the caudate lobe with 7-0 silk were performed. The same procedure was then repeated on branches of the portal veins supplying the right and left lobes. Subsequent dissection and ligation of the right branch of the portal vein which supplied the median lobe was followed by a line of demarcation between the perfused and the unperfused parts of the median lobe. To complete Step I of the ALPPS procedure, transection of liver parenchyma along the demarcation line was carried out using a disposable drip bipolar coagulation forceps down to the anterior aspect of the inferior vena cava. In the PVL group, the rats were operated in the same way as the rats in the ALPPS group, with the exception of not carrying out transection of hepatic parenchyma along the demarcation line. The procedure of PVL was performed as shown in The rats in all the groups underwent a relaparotomy 72 hours after Step 1. The caudate lobe, right lobe and right median lobe were resected, keeping only the left median lobe. The rats in each group were sacrificed on day 1, 2, 3, 5 and 7 after the second operation (n = 4 to 6 at each time point). Blood samples were collected from the inferior vena cava at each time point. They were centrifuged at 3000 g for 10 min, and the serum was stored at −80 °C until the tests for liver biochemical determinations were performed. After weighing, approximately 200 mg of liver tissues from the right and left median lobes were immediately frozen in liquid nitrogen and then stored at −80 °C. The remaining liver lobes were fixed in formaldehyde.Total RNA was extracted from 50 mg of liver tissues using TRIzol reagent . Five micrograms of RNA were reverse-transcribed using the Thermoscript RT-PCR kit (Invitrogen), yielding the complementary DNA template. The quantitative RT-PCR amplification and the data analysis were performed using an ABI-Prism 7000 Sequence Detector system. The primers for IL-6, HGF, and TNF-α areshown in Regenerating liver samples were homogenized in buffer  and centrifuged , and the supernatant was used for analysis. The concentrations of proteins in the liver tissues were measured with ELISA kits, IL-6 , TGF-α , NF-KB , STAT3 , TNF-α, OSCF , HGF , JAK , SOCS-3 , ERK1/2 , c-met , Src , Akt , c-myc , c-jun  and YAP  according to the manufacturer’s instructions.The blood samples obtained from the inferior vena cava at different time points were immediately centrifuged at 2000 g for 5 minutes. Serum aspartate aminotransferase (AST) and alanineaminotransferase (ALT) levels were measured with a commercial assay kit  using a serum Hitachi 7020 automatic biochemical analyzer. Enzyme activities were expressed as an international unit per liter (U/L).The liver tissues were immersion fixed in 4% formaldehyde, embedded, sectioned, and stained with hematoxylin-eosin (H&E) for the necrotic area determination. The liver sections were immunostained for Ki-67  or proliferating cell nuclear antigen (PCNA)  as described previouslyStandard western blot assays were used to analyze protein expression, as described previouslyImages were acquired and analyzed as described previouslyThe detailed methodology has been described previouslyImmunoprecipitation and histone H1 kinase assay (Cdk2 activity) were performed as previously describedP was smaller than 0.05. Statistics were performed using the software package GraphPad 4.0  and SPSS 12.0.1 .All data are expressed as means ± standard deviation (SD). Differences between the groups were assessed by one-way or two-way analysis of variance , using an appropriate post-hoc comparison test. A significant difference was assumed when How to cite this article: Shi, H. et al. A preliminary study of ALPPS procedure in a rat model. Sci. Rep.5, 17567; doi: 10.1038/srep17567 (2015).


Pre-eclampsia or Eclampsia (PE or E) accounts for 25% of cases of maternal mortality worldwide. There is some evidence of a link to dietary factors, but few studies have explored this association in developing countries, where the majority of the burden falls. We examined the association between adequately diversified dietary intake, iron and folic acid supplementation during pregnancy and symptoms suggestive of PE or E in Indian women.Cross-sectional data from India’s third National Family Health Survey  was used for this study. Self-reported symptoms suggestive of PE or E during pregnancy were obtained from 39,657 women aged 15-49 years who had had a live birth in the five years preceding the survey. Multivariable logistic regression analysis was used to estimate the association between adequately diversified dietary intake, iron and folic acid supplementation during pregnancy and symptoms suggestive of PE or E after adjusting for maternal, health and lifestyle factors, and socio-demographic characteristics of the mother.In their most recent pregnancy, 1.2% (n=456) of the study sample experienced symptoms suggestive of PE or E. Mothers who consumed an adequately diversified diet were 34% less likely  to report PE or E symptoms than mothers with inadequately diversified dietary intake. The likelihood of reporting PE or E symptoms was also 36% lower  among those mothers who consumed iron and folic acid supplementation for at least 90 days during their last pregnancy. As a sensitivity analysis, we stratified our models sequentially by education, wealth, antenatal care visits, birth interval, and parity. Our results remained largely unchanged: both adequately diversified dietary intake and iron and folic acid supplementation during pregnancy were associated with a reduced occurrence of PE or E symptoms.Having a adequately diversified dietary intake and iron and folic acid supplementation in pregnancy was associated with a reduced occurrence of symptoms suggestive of PE or E in Indian women. Eclampsregnancy . Pre-eclregnancy , and is regnancy ,5. Basedregnancy ; clinicaregnancy –9. HowevAlthough the etiology of PE or E remains unclear, the role of maternal diet in the development of PE or E has recently received increased attention –11. SeveSeveral epidemiologic studies indicate that consumption of fruits, vegetables, and dietary fibre is associated with lower pre-eclampsia risk –26, possAssociations between diversified dietary intake, micronutrients, and hypertensive disorders are particularly relevant in India, where rates of malnutrition and micronutrient deficiencies are high among pregnant women . To our Identifying the link between an adequately diversified dietary intake and the risk of PE or E may suggest an important point of intervention at both the peri-conceptional and gestational stages particularly in a developing country such as India. In this study, as dietary diversity is a key factor in ensuring an adequate micronutrient balance, we use the large-scale, cross-sectional and nationally representative third National Family and Health Survey (NFHS-3) data –38 to teCross-sectional data from the most recent wave of the National Family Health Survey , India’s Demographic and Health Survey (DHS), was used for this study. These data are publicly available by request from DHS . The surTo assess symptoms of PE or E, we restricted the sample to those women who had a live birth in the five years preceding the survey. We further restricted our analyses to data pertaining to the most recent birth, both to minimize recall bias and in order to draw on iron and folic acid supplementation and antenatal care (ANC) measures, which were only available for only the most recent pregnancy. This resulted in a final sample size of 39,657 participants.To assess the occurrence of PE or E, we constructed a measure based on women’s self-reports of symptoms during pregnancy. Specifically, mothers were asked: “During this pregnancy, did you have difficulty with your vision during daylight?”, “During this pregnancy, did you have swelling of the legs, body or face?”, and “During this pregnancy, did you have convulsions not from fever?” The response options were “Yes”, “No”, and “Don’t know”. Following the World Health Organisations  and NatiDietary diversity is often used as a proxy for dietary intake, both because it is a straightforward measure of nutrition, and also because the burden on respondents is relatively low . The WHOThe other main exposure variable used in our study was self-reported consumption of iron and folic acid supplementation during pregnancy. For the most recent birth, NFHS-3 asked to mothers “Were you given or did you buy any iron and folic acid tablets or syrup?” In addition, the duration (in days) of iron and folic acid supplementation was recorded, and was categorized based on proposed national guidelines and WHO guidelines . As ironGiven the unknown etiology of PE or E, it is possible that its onset is influenced by sociodemographic and health-related factors. In order to adjust for this, we controlled for three groups of potential confounders.Categorical measures of age  and birth interval  and a dichotomous indicator of pregnancy type  were included to control for the positive association between maternal age –45, shor2) measure, categorized as underweight (≤18.5 kg/m2), normal (18.5 to 22.9 kg/m2), overweight (23.0 to 24.9 kg/m2), and obese (≥25.0 kg/m2). As change in BMI between pregnancies has been estimated to be over one-half a unit  [As dietary patterns are likely to be part of a broader health lifestyle, which may itself have implications for PE or E risk, we included a group of covariates to adjust for health and lifestyle factors. Studies have demonstrated that women with pre-eclampsia have a higher rate of pre-gestational hypertension and overweight or obese body mass index (BMI) . Followi0.3–1.7) , we assu0.3–1.7) –59. In N0.3–1.7) –62, we c0.3–1.7) –64 and t0.3–1.7) –66. The 0.3–1.7) –68. Some0.3–1.7) . We thusIn order to reduce the risk of unobserved homogeneity in our models, we included a variety of socio-demographic controls. Religion was categorized into Hindu, Muslim, Christian, Sikhs or other/missing . Caste was categorized as scheduled caste, scheduled tribe, other backward class , general, and other/missing . We also2 tests. We assessed the possibility of multicollinearity between the covariates by using a correlation matrix of covariates. All pairwise Pearson correlation coefficients were less than 0.5, suggesting that multicollinearity is not a concern. We included NFHS-3 sampling weights to account for the sample design and ensure the representativeness of the sample [All analyses were conducted using the SPSS statistical software package Version 19 . Standard descriptive statistics were calculated for all variables, including means and standard deviations. Differences in categorical variables were tested using χe sample . Fixed ee sample –71 were Multivariable models were adjusted for the above described maternal factors, health and lifestyle factors and socio-demographic characteristics of the mother. In the first logistic regression model, we examined the unadjusted association between dietary diversity, iron and folic acid supplementation and PE or E symptoms independent of each other. In the second model, we adjusted for maternal factors in order to assess how much of the variance in this association was explained by maternal characteristics. In the third model, we added health lifestyle factors to our model. In the fourth and final model, we added socio-demographic characteristics in order to examine the association between dietary diversity, iron and folic acid supplementation and PE or E symptoms controlling for all the confounders discussed above. Maternal marital status was not included as part of the socio-demographic characteristics because the majority  were married. We also tested for a variety of potential interactive effects, for any possible significant interactions. In particular, given that ANC visits may improve maternal health outcomes both by direct treatment of health conditions and by providing women with dietary advice and supplementation, we tested for an interaction between ANC visits with each of our two key predictors. State fixed effects were included in all models, and standard errors were clustered at the primary sampling unit.It has been argued that logistic regression models should be used with a minimum of 10 events per predictor variable (EPV) –74. In tWomen with better socioeconomic resources may a) have a better understanding of their symptoms, b) report their experiences differently when compared to women in lower wealth and education groups, and c) have access to a better diet during pregnancy. To overcome potential sources of information bias, particularly with regard to self-reported diet and health problems during pregnancy, we ran sensitivity analyses for level of education (higher than secondary) and household wealth (richest quintile). Given that the population of women attending ANC might be inherently different than those who do not, we also conducted a sensitivity analysis on ANC visits, restricting the sample to only those women who attended ANC.Women who were currently pregnant or who had given birth within 2 months of the survey were not weighed, resulting in missing values on the BMI measure for currently or recently pregnant women. Due to list wise deletion, these women were excluded from our full regression models. Given that pregnancy should in theory be randomly distributed in the sample, it is unlikely that this sample restriction would bias our sample. However, to test for potential bias, we created a variable to flag cases that were missing on BMI, and ran our fully adjusted model including the flag variable, but without BMI as a covariate in order to retain these cases. However, since BMI is an important covariate that could influence the effect estimate, we have again re-estimated the models by adding an additional category to the existing BMI variable to signify those who were missing on the measure.We also repeated the analysis, restricting it to those women who had given birth up to 3 years prior to the survey and reported PE or E symptoms to examine the potential effects of recall bias. Short birth intervals are associated with adverse perinatal outcomes , but lonApproximately one-fifth (18.5%) of the mothers reported a spontaneous or induced abortion. Malnutrition rates were high: almost one-fifth of the mothers were moderately or severely anaemic, 31% were underweight while 22% were either overweight or obese. More than half the mothers had four or more ANC visits during their last pregnancy and almost 70% had access to the private medical sector to obtain their health care. Very few were current smokers (1.5%) or alcohol drinkers (2.3%), while almost one in ten were smokeless tobacco users (8.3%). Prevalence of diabetes (1.3%) and asthma (1.2%) were low. Most mothers  were aged 15–29 years, and almost half (47.4%) had no education. A majority of the mothers (four out of five) were identified as Hindu, and two-fifths belonged to a disadvantaged class. One in four mothers were agricultural worker while a very few (1.8%) worked in the service sector. One fourth belonged to the household with poorest wealth. More than 70% of the mothers were residing in rural areas and 28% were residents of Central India.Considering prevalence of PE or E, a higher proportion of women with inadequate dietary diversity reported of PE or E symptoms (1.4%) than those women with adequately diversified dietary intake (0.6%). A higher proportion of those who did not consume iron and folic acid supplementation for at least 90 days also reported of high PE or E (1.3%) symptoms as compared to those who took iron and folic acid supplements for 90 or more days (0.6%). Women with mild (1.3%) and moderate anaemia (1.5%) also reported higher symptoms of PE or E as compared to those with no (0.9%) or severe (0.9%) anaemia. Compared to overweight (0.8%) or obese (0.5%) women, those who were underweight (1.3%) and normal weight (1.3%) reported higher proportion of PE or E symptoms.Pregnancy and health risk factors were also significantly associated with PE or E symptoms, with a higher proportion of women of parity 4 or higher (1.7%) reported higher PE or E symptoms than women of lower parity. First order births had the lowest proportion of PE or E symptoms (0.8%), while women with a birth interval of 3 or more years experienced significantly higher symptoms of PE or E (1.4%). Multiple births were also associated with higher odds of PE or E symptoms (3.1%) compared to singleton births (1.1%), as were previously aborted pregnancies (1.4%) compared to those with no previous spontaneous or induced abortions (1.1%). A higher proportion of current smokeless tobacco users (1.8%), current alcohol drinkers (2.6%), and current tobacco smokers (1.7%) also reported PE or E symptoms compared to those who, respectively, do not currently use tobacco, drink, or smoke. Additionally, asthmatics (4.7%) reported higher PE or E symptoms than non-asthmatics (1.1%).Significant associations were also found between socio-demographic characteristics and PE or E symptoms. A lower proportion of women aged 15–29 reported PE or E symptoms (1.1%) compared to women aged 30–39 (1.4%) and 40–49 years (1.3%). However, a higher proportion of women with no education (1.6%) reported PE or E symptoms compared to those with primary, secondary, or higher levels of education. Caste was also a significant correlate, with a higher proportion of women reporting PE or E symptoms belonging to a scheduled tribe (2.2%) than those in a scheduled caste (1.2%), disadvantaged class (1.1%) or general caste (0.8%). Those in the poorest wealth quintile reported the highest PE or E symptoms (2.0%) compared to those in the other four wealth quintiles, as did those in professional (1.2%) and agricultural (1.7%) occupations. Rural residence was also associated with increased proportion of women reporting a PE or E symptoms (1.4%) compared to urban residence (0.6%), and women living in central (1.7%), northeast (1.4%) and eastern (1.4%) India reported higher PE or E symptoms than those in other regions.not shown on the table). We again re-estimated the models with BMI split into 5 categories  and could found no demonstrable change in the association. Among women in the highest education group, the likelihood of reporting symptoms of PE or E was lower among those with a adequately diversified dietary intake  and recommended intake of iron and folic acid supplementation  during pregnancy (not shown on the table). Even after additional adjustment for ANC visits, intake of an adequately diversified diet was associated with a 43%  and iron and folic acid supplementation was associated with a 41% reduction in the likelihood of PE or E  symptoms. Restricting our analysis to those women who had given birth up to 3 years prior to the survey and reported PE or E symptoms (1.4%), we found that the association of adequately diversified dietary intake, iron and folic acid supplementation, and PE or E symptoms was non-significant. However, the number of women in this restricted sample was very small (n = 46), and as a result the models had limited statistical power. In a final set of sensitivity analyses restricting the models to multiparous women we found that the significant inverse association between diversified dietary intake, intake of iron and folic acid supplementation during pregnancy and symptoms of PE or E still remained.As a robustness check, we ran Models 1 through 4 restricting only to those cases with valid data on the BMI measure. Results remained largely unchanged: diversified dietary intake  and intake of iron and folic acid supplementation  continued to be significant predictors of lower occurrence of PE or E symptoms in Indian women  were self-reported, including a symptomatic rather than clinical measure of PE or E; it is possible that self-reported data may suffer from recall bias. Although we cannot rule out the possibility of misclassification within this context, it is unlikely that we have missed severe PE or E cases due to the generally clear manifestation of symptoms in severe cases. Second, due to the nature of the data, we could not identify the gestational onset of PE or E; it is possible that nutritional factors may be particularly important during critical periods of foetal development, which we are not able to assess in this study. Third, the role of folic acid independent of iron supplementation may be of interest. However, because iron and folic acid were measured jointly rather than as separate supplements in NFHS-3, we were unable to disentangle the effects of these two supplements. Similarly, information about other micronutrient supplementation is unavailable in the NFHS-3; while our models control for a wide range of health and sociodemographic factors, it is possible that other micronutrients, such as B12, may also shape PE or E risk. Further research is needed at the population level to understand the independent effects of micronutrient supplementation. An additional concern regarding the self-reported data is potential misreporting of dietary intake; this misreporting would most likely be non-differential, and may attenuate the association between dietary diversity and PE or E symptoms. Moreover, the broad categories of food intake in the NFHS-3 limits our ability to assess the impact of particular food items, especially those associated with diabetes (such as sugar) and other health conditions. An adequately diversified dietary intake was associated with higher socioeconomic status in our study; higher socioeconomic status may be associated with both better diets and regular health check-ups. However, the results of our sensitivity analyses of models stratified by wealth, education, and ANC visits suggest that this bias is limited.both difficulties with daytime vision and swelling of the face and extremities during pregnancy. This more stringent definition may result in a conservative bias in our results, as some women who reported experiencing only one symptom (and thus were coded as not experiencing PE or E here) may have been identified as pre-eclamptic via clinical diagnosis.In our sample, the prevalence of PE or E symptoms was quite low (1.2%) as compared to other studies in India. In part, this low prevalence is likely reflects the fact that survey participants were selected from the general population rather than from the higher-risk populations used in many clinical and epidemiological studies. Additionally, because we were unable to confirm PE or E with a clinical diagnosis, we have used a particularly stringent measure, requiring women to have experienced Our study also showed the prevalence of PE or E was higher among women of higher parity, lean women, current smokers, and the less-educated. While these findings are in contrast to some of the existing literature conducted in developed countries, they are in keeping with other work among Asian women ,98–99. OAdverse maternal and infant health outcomes often can be prevented by modifying maternal behaviours . MonitorWith the target of the Millennium Development Goals and adherence to India’s Five Year Plan for reproductive health in sight, PE or E should be identified as a priority area in reducing maternal morbidity in India. Dietary diversity and nutritional adequacy in pregnancy represent modifiable risk factors for PE or E, and should be considered as targets for prevention. Efforts which exclusively target micronutrient supplementation may not be sufficient for reducing PE or E symptoms. Policies aiming to reduce the risk of hypertension in pregnancy should target improving access to, and compliance with, the simple recommended therapy of iron and folic acid supplementation and an adequately diversified dietary intake in tandem. However, as this analysis has demonstrated, there may be a substantial socioeconomic gradient in health risk behaviour and access to preventive care. Programmatic interventions should focus particularly on women with limited socioeconomic resources. For example, programs targeting food security among low income groups in India, such as the Public Distribution System (PDS), could improve maternal and child health and reduce the risk of PE or E by offering a more diversified basket of subsidized food items.Using nationally representative data on reproductive health among Indian women, our study shows that having an adequately diversified dietary intake and iron and folic acid supplementation in pregnancy is associated with a reduced occurrence of symptoms suggestive of PE or E in Indian women. This relationship persists when sociodemographic, health lifestyle, and maternal factors are taken into consideration. Because ANC is the main source of iron and folic acid supplements and nutrition education during pregnancy, ensuring universal provision of comprehensive ANC, emphasized in India’s Eleventh Five Year Plan (2007–2012) , is vita


Land use change alters the structure and composition of microbial communities. However, the links between environmental factors and microbial functions are not well understood. Here we interrogated the functional structure of soil microbial communities across different land uses. In a multivariate regression tree analysis of soil physicochemical properties and genes detected by functional microarrays, the main factor that explained the different microbial community functional structures was C : N ratio. C : N ratio showed a significant positive correlation with clay and soil pH. Fields with low C : N ratio had an overrepresentation of genes for carbon degradation, carbon fixation, metal reductase, and organic remediation categories, while fields with high C : N ratio had an overrepresentation of genes encoding dissimilatory sulfate reductase, methane oxidation, nitrification, and nitrogen fixation. The most abundant genes related to carbon degradation comprised bacterial and fungal cellulases; bacterial and fungal chitinases; fungal laccases; and bacterial, fungal, and oomycete polygalacturonases. The high number of genes related to organic remediation was probably driven by high phosphate content, while the high number of genes for nitrification was probably explained by high total nitrogen content. The functional gene diversity found in different soils did not group the sites accordingly to land management. Rather, the soil factors, C : N ratio, phosphate, and total N, were the main factors driving the differences in functional genes across the fields examined. Nutrient cycling within terrestrial ecosystems is mostly performed via the activities of soil-borne microorganisms . With thA large amount of knowledge is becoming available concerning the microbial enzymes responsible for the key steps of the major nutrient cycles in soil . Recent studies have revealed a great diversity within the genes encoding these key enzymatic processes –10, provAnthropogenic perturbations  are known to influence soil nutrient cycles, but little is known about the mechanistic aspects of such disturbances. This lack of knowledge inhibits our ability to assess the extent to which human activities disturb terrestrial nutrient cycling potential and thwarts efforts to predict future anthropogenic impacts. Before the influence of such perturbations can be established or predicted, one must characterize the natural variation and normal operating range with respect to the diversity and expression of key genes related to nutrient cycle functions. To establish such normal operating boundaries, the dynamics of the gene diversity and expression must be monitored across relevant spatial and temporal scales and in response to natural and imposed variability. Although this still remains technically challenging, with hurdles related to limits of signal detection and reproducibility , 16, funIn the current study, we exploited the functional structure of microbial communities in soils under different land uses. First, we determined the soil parameters of each field. Next, we tracked the microbial functional communities across land uses. Lastly, we identified links between soil parameters and microbial functions. The main questions addressed in this study were how does the microbial community functional structure vary with different soil managements and what are the main drivers that are related to this variation. In order to answer these questions, we used functional gene arrays (FGA), focussing on crucial steps of key nutrient cycles  across eight fields representing five generally representative forms of land use  in The Netherlands. To our knowledge, this is one of few studies to apply functional microarray technique across a range of different land managements. In addition, this study is one of the first to include a suite of soil measurements to explore the environmental factors driving soil microbial functions under different land uses.http://dx.doi.org/10.1155/2014/216071) in May 2007; see Eight fields subjected to five generally representative forms of land management in The Netherlands  were sampled . Soil pH was measured in a 1 : 2.5 soil/water suspension, and soil moisture was determined gravimetrically (g/100 g). Soil organic matter content (% OM) was determined by loss on ignition (LOI) analysis. Soil texture was determined using a Bouyoucos densimeter after shaking the soil vigorously with NaOH 1 M as dispersant. Soil CaCO3 was determined a by Scheibler's method. Phosphate (P) was determined as the amount extracted from a soil after addition of water at a shaking ratio of 1 : 60, a procedure typically used to determine the soil fertility status of arable fields in The Netherlands. Cr, Cu, Hg, As, Ni, Cd, Pb, and Zn were extracted by Mehlich 1 and determined by atomic absorption spectrometry.Physicochemical characterization was performed by BLGG .DNA extractions were performed separately on each of the five replicates per field using the MoBio Power Soil Extraction kit  with bead-beating  at 5.5 m sThe DNAs from replicates of each sample were pooled. Because the amount of DNA extracted was in most cases insufficient for direct labeling and hybridization, 30 ng of DNA per sample was amplified by whole community rolling cycle amplification (WCRCA) using a TempliPhi kit . We used the GeoChip functional microarray platform, which contains more than 24,000 oligonucleotide (50-mer) probes targeting targeted approximately 10,000 genes involved in nitrogen, carbon, sulfur and phosphorus transformations and cycling, metal reduction and resistance and organic xenobiotic degradation . The numThe data processing was according to He et al. . Each ar3, Cr, Cu, P, Zn, soil texture, and soil moisture) using the “multtest” package in R . P values were corrected for multiple testing, using the false discovery rate controlling procedure [Pearson correlations were calculated between soil factors  in Canoco 4.5 for Windows . Probe i3 was found in organic arable field 13F. The soil moisture at the time of sampling varied among fields. The moisture contents of 19F and 25F fields were the highest, while moisture contents of 10F and 1F fields were the lowest .A total of 1405 genes across all samples were detected. The pasture field 1F had the lowest numbers of genes (146) detected by the GeoChip followed by conventional arable field 8F (173 genes) and natural grassland field 25F . The orgIn general, there were differences in microbial community functional structure among the study sites . The per3, Cd, Cr, Cu, Ni, Pb, Zn, As, and Hg) and the intensities of the 1405 genes detected by the GeoChip, the main factor that explained the microbial community functional structure differences in the eight fields was C : N ratio (In a multivariate regression tree (MRT) analysis, combining the measured soil factors  and C/D  were that fields with C : N ratios lower than 11 had more genes for carbon degradation (CDEG), carbon fixation (CFIX), metal reductase (MET), and organic remediation (ORG) categories than the fields with C : N ratio higher than 11. C : N ratio is known to have a large influence on decomposition rates . Fields on genes  in fieldAmong fields with C : N ratio lower than 11, field 16F had the highest number of genes of the organic remediation (ORG) category. This may be related to the high amount of phosphate in this soil—that is, greater than 107 mg/kg soil as calculated in MRT analysis and illustrated in the CCA plot . On the The genes detected within the ORG category in 16F are related to a range of degradation activities Table S2). In fact, phosphate has been shown to be an important nutrient factor required by bacterial biofilter for maximum methane elimination . The fie. In fact amoA, pmoA, and ghd. The DSR genes abundant in 8F were dsrA and dsrB; the methanol-oxidation category was dominated by pmoA and mmoA from methanotrophs and nitrogen fixation genes categorized as nifH from nitrogen-fixing bacteria (Table S4).Fields with a C : N ratio higher than 11  had relatively high percentages of genes of nitrification (NIT), and high levels of total nitrogen  appeared to explain this result. The field 8F had the highest numbers of genes from categories for dissimilatory sulfate reductase, methane oxidation, and nitrogen fixation, and this pattern was explained by total N levels lower than 1302 mg/kg soil . The higβ-Proteobacteria amoA genes. Ureases (E.C. 3.5.1.5) are complex metalloenzymes that catalyze the hydrolysis of urea to ammonia and carbon dioxide. This enzyme allows many soil bacteria to use urea as nitrogen source, and we detected several ureases in nitrogen-fixing bacteria such as Mesorhizobium loti, Bradyrhizobium japonicus, Rhodopseudomonas palustris, Rhodobacter sphaeroides, Rhodobacter (α-Proteobacteria), Chromobacterium violaceum (β-Proteobacteria), and Nostoc (Cyanobacteria) (Table S4). Urease is also an important virulence factor that improves survival of pathogenic bacteria in harsh conditions within the host and causes direct damage to the host due to ammonium, CO2, or alkali production , Mycobacterium (Actinobacteria), and Brucella melitensis (α-Proteobacteria) (Table S4).The nitrification genes found in fields 1F, 4F, 10F, and 25F were similar to uncultured ammonia-oxidizing iews see , 28), anFields 4F and 25F had distinct soil properties or conditions that would be expected to impact numerous microbial processes. However, we found no evidence for a sharp distinction in the functional gene repertoires of these communities. Field 4F was extremely acidic which distinguishes it from other natural and cultivated soils. In fact, in a previous study on microbial community composition across 26 fields under different land uses including the eight fields here studied, Kuramae et al.  found thThe soil-borne microbial functional structure in the different fields in The Netherlands did not group the sites accordingly to land management. Although the number of fields examined here was limited to eight, the breadth of our study was sufficient to assess the differences in microbial functional genes in different systems of soil management, and specific soil factors could be identified that explained the differences observed in functional gene composition of the different soils examined.Supplementary material is regarding to soil physicochemical factors, genes of organic remediation, carbon degradation, metal reductase and nitrogen fixation categories overrepresented in diffrent fields.


Total crop production of each county was significantly, positively related to SOC, TSN, soil C and N store, and soil C and N stock. Soil C and N were positively correlated with soil bulk density but negatively correlated with soil porosity. These results indicate that variations in soil C could regulate crop yield in intensive agricultural systems, and that spatial patterns of C and N levels in soils may be regulated by both climatic factors and agro-ecosystem management. When developing suitable management programs, the importance of soil C and N stores and their effects on crop yield should be considered.Improved management of soil carbon (C) and nitrogen (N) storage in agro-ecosystems represents an important strategy for ensuring food security and sustainable agricultural development in China. Accurate estimates of the distribution of soil C and N stores and their relationship to crop yield are crucial to developing appropriate cropland management policies. The current study examined the spatial variation of soil organic C (SOC), total soil N (TSN), and associated variables in the surface layer (0–40 cm) of soils from intensive agricultural systems in 19 counties within Henan Province, China, and compared these patterns with crop yield. Mean soil C and N concentrations were 14.9 g kg Safeguarding food security and ensuring sustainable development are two fundamental goals of intensive agriculture in China Indeed, variations in soil C and N stores may closely regulate crop yield, although published data on the relationship between these parameters are inconsistent. Some studies have reported a positive correlation between soil C and N and crop yield Soil C and N stores in crop lands, especially in the topsoil layer, are potentially greatly affected by human activity; thus, understanding the spatial pattern of soil C and N stores on a regional scale is crucial to developing a management strategy for improving soil fertility −1 annually has been applied in this production area, resulting in an increase in soil acidity Henan Province is the second largest area of crop production in China . To produce an adequate supply of food for the domestic population, unsustainable production methods have often been used in this province. Historically, intensive production based on an annual wheat-maize system has been used to achieve high crop yield. This practice, however, has resulted in badly degraded agricultural soils, causing erosion and a loss of good soil structure. More than 600 kg N haThe objective of the present study was to characterize the spatial distribution of C and N stores in intensively cultivated counties within the Henan Province of China and to determine the relationship between crop yield and soil organic C and N.Statement: We have field permits for sampling soil in each of the field sites within each county of Henan Province, China. All of the sampling sites are privately owned, and there was no potential impact on any endangered or protected species among these sampling sites.http://ngcc.sbsm.gov.cn/) using ArcGIS software. As of 2009, the human population of Henan was about 9.9×107 persons. The Province is approximately 167,000 km2 in land area, lying within the monsoonal temperate zone. It has a cultivated land area of 79, 260 km2 for the production of wheat and maize. There are three dominant soil types in Henan Province: Yellow-cinnamon soil (Eutric Cambisols in FAO taxonomy), Sajiang black soil (Eutric Vertisols/Gleyic Cambisol), and Fluvo-aquic soil (Fluvisols in FAO taxonomy) The study was carried out in 19 counties within Henan Province, located in central China . Map dathttp://www.ha.stats.gov.cn/hntj/index.htm). Annual yield data for winter wheat and total crop production in 2009 were also obtained from Henan Statistical Yearbook 2010 (29-7) and the Agricultural Bureau of each of the 19 counties in which soil sampling took place  and wheat yield from 1978–2009  were obtok place . These cThe 19 counties were selected as representative of the main agro-ecosystems of Henan Province. Soil samples were collected during June 1–15, 2009 following the wheat harvest but prior to the sowing of maize. Six representative, replicate field plots, located at least 6 km apart, were selected within each county based on four criteria: (1) the field plots had been continuously cultivated for at least 30 yr with a native variety, (2) the cropland area was located within 5 km of native vegetation with a similar landscape, soil type and texture, and a relatively flat terrain, and (3) all of the sampling sites are privately owned, and (4) there was no potential impact on any endangered or protected species in the sampling site. Geographic coordinates of each sampling site was recorded by handed GPS of Magellan eXplorist 210(USA), and all of these data were attached in the supporting information.2 were established in each plot, with sixteen sampling points taken at random in each of two layers (0–20 cm and 20–40 cm) using a 70 mm - diameter auger. All of the soil samples taken at each layer within a sample plot were mixed together and treated as one sample to represent the value of the plot, yielding 114 soil samples at each layer.Sample areas of ∼1300 m2Cr2O7 and 10 mL of concentrated H2SO4 at 150°C, followed by titration of the digests with standardized FeSO4. Total soil N (TSN) was measured using a modified Kjeldahl wet digestion procedure and a Tector Kjeltec System 1026 distilling unit. Soil available N was analyzed using a micro-diffusion technique after alkaline hydrolysis (1.8 mol L−1 NaOH). The Olsen method was used to determine available soil phosphorus (P), and available soil potassium (K) was measured in 1 mol L−1 NH4OAc extracts by flame photometry  was measured using a modified Mebius method. Briefly, 0.1 g soil samples were digested for 5 min with 5 mL of 1N Kotometry .3 soil cores obtained from the four layers. Soil porosity was calculated from soil bulk density and specific gravity, with any stone material removed and not considered in bulk density calculations.Three sampling points were used to determine soil bulk density in each plot. Samples were collected separately from four layers within a depth of 0–40 cm in each sampling point. Soil bulk density was measured using 100-cmTotal soil organic C store (TSOCS) and total soil N stores (TSNS) at 0–40 cm depth were calculated as follows:area is the given total cultivated area of each county, and CS and NS are C and N stocks, respectively. SOC and TSN were means of six sampling sites of each county.Given the cultivated area, the total cultivated topsoil (0–40 cm) C and N stocks of each county were estimated by the equation:α = 0.05. Linear regression was used to determine the relationships between C and N stock versus wheat and total crop production. Principle components analysis was used to assess patterns of similarity/dissimilarity among counties with respect to several environmental variables Analysis of variance was used to assess the significance of location (county) on soil C and N concentration and storage; means were compared using Duncan’s multi-range test at Wheat yields increased more than 250% from 1978 to 2009 while total annual crop production in Henan Province increased from 21 to 54 million tons over the same time period . Wheat y−1 among the 19 counties in 2009  varied from 221 to 659 g m−2. The highest value was in XX County and the lowest in LY County in N reserves , soil C  and N store . While soil porosity was significantly and negatively correlated with soil N concentration , soil C  and N store .Linear regression analysis indicated that total crop production was significantly and positively correlated with SOC and TSN , soil C Principle components analysis revealed that Axis 1, which explained 98% of the variation in all data , was highly correlated with soil C, whereas Axis 2, explaining 1% of the variation , was highly correlated with soil N. Thus, counties such as QX and XP located highly positive on Axis 1 with high levels of soil C, but other counties, such as LK, YJ, and QF, occupied positions toward the negative end of Axis 1 with low soil C .−1 and 10 to 300 kg ha−1 for wheat and maize, respectively, following increases of 1 MT of C in agricultural soils in Africa It is notable that 14 environmental  toward the lower end and XP and QX (mean soil C = 20.5 g C kg−1) toward the upper end of Axis 1, which accounted for nearly 80% of the variation in soil and environmental data  located toward the lower end of Axis 2 (accounting for <10% of variation) and XX and TY (mean soil N = 1.81 g N kg−1) located toward the upper end of Axis 2 (Spatial variation in soil N was essentially orthogonal to that of soil C. This was surprising since typically, the two are highly correlated in terrestrial ecosystems f Axis 2 . AlthougManagement methods used in crop production systems, including tillage practices and fertilizer use, can affect soil C and N on broad spatial scales, including that of an entire Province Results presented in the current study underscore the complexity of factors that can impact agricultural soils and their ability to produce crops to meet the ever-increasing demand in China resulting from population growth. Some of the spatial pattern exhibited in ordination space  is clear