Assessment of a non-invasive approach to pregnancy diagnosis in gray whales through drone-based photogrammetry and faecal hormone analysis.

Knowledge of baleen whales' reproductive physiology is limited and requires long-term individual-based studies and innovative tools. We used 6 years of individual-level data on the Pacific Coast Feeding Group gray whales to evaluate the utility of faecal progesterone immunoassays and drone-based photogrammetry for pregnancy diagnosis. We explored the variability in faecal progesterone metabolites and body morphology relative to observed reproductive status and estimated the pregnancy probability for mature females of unknown reproductive status using normal mixture models. Individual females had higher faecal progesterone concentrations when pregnant than when presumed non-pregnant. Yet, at the population level, high overlap and variability in progesterone metabolite concentrations occurred between pregnant and non-pregnant groups, limiting this metric for accurate pregnancy diagnosis in gray whales. Alternatively, body width at 50% of the total body length (W50) correctly discriminated pregnant from non-pregnant females at individual and population levels, with high accuracy. Application of the model using W50 metric to mature females of unknown pregnancy status identified eight additional pregnancies with high confidence. Our findings highlight the utility of drone-based photogrammetry to non-invasively diagnose pregnancy in this group of gray whales, and the potential for improved data on reproductive rates for population management of baleen whales generally.

Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae.

Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats. These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer. In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences. We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences. We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence. Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates. These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.

Regulation of the MEF2 family of transcription factors by p38.

Members of the MEF2 family of transcription factors bind as homo- and heterodimers to the MEF2 site found in the promoter regions of numerous muscle-specific, growth- or stress-induced genes. We showed previously that the transactivation activity of MEF2C is stimulated by p38 mitogen-activated protein (MAP) kinase. In this study, we examined the potential role of the p38 MAP kinase pathway in regulating the other MEF2 family members. We found that MEF2A, but not MEF2B or MEF2D, is a substrate for p38. Among the four p38 group members, p38 is the most potent kinase for MEF2A. Threonines 312 and 319 within the transcription activation domain of MEF2A are the regulatory sites phosphorylated by p38. Phosphorylation of MEF2A in a MEF2A-MEF2D heterodimer enhances MEF2-dependent gene expression. These results demonstrate that the MAP kinase signaling pathway can discriminate between different MEF2 isoforms and can regulate MEF2-dependent genes through posttranslational activation of preexisting MEF2 protein.

Mismatch correction acts as a barrier to homeologous recombination in Saccharomyces cerevisiae.

A homeologous mitotic recombination assay was used to test the role of Saccharomyces cerevisiae mismatch repair genes PMS1, MSH2 and MSH3 on recombination fidelity. A homeologous gene pair consisting of S. cerevisiae SPT15 and its S. pombe homolog were present as a direct repeat on chromosome V, with the exogenous S. pombe sequences inserted either upstream or downstream of the endogenous S. cerevisiae gene. Each gene carried a different inactivating mutation, rendering the starting strain Spt15-. Recombinants that regenerated SPT15 function were scored after nonselective growth of the cells. In strains wild type for mismatch repair, homeologous recombination was depressed 150- to 180-fold relative to homologous controls, indicating that recombination between diverged sequences is inhibited. In one orientation of the homeologous gene pair, msh2 or msh3 mutations resulted in 17- and 9.6-fold elevations in recombination and the msh2 msh3 double mutant exhibited an 43-fold increase, implying that each MSH gene can function independently in trans to prevent homeologous recombination. Homologous recombination was not significantly affected by the msh mutations. In the other orientation, only msh2 strains were elevated (12-fold) for homeologous recombination. A mutation in MSH3 did not affect the rate of recombination in this orientation. Surprisingly, a pms1 deletion mutant did not exhibit elevated homeologous recombination.

The p38 MAP kinase pathway and its biological function.

p38 is a mitogen-activated protein (MAP) kinase with structural and functional characteristics that distinguish it from JNK and ERK MAP kinases. p38 activity is upregulated when cells are exposed to a variety of stimuli including bacterial pathogens, proinflammatory cytokines, certain growth factors, and other forms of environmental stress. By regulating downstream substrates that include protein kinases and transcription factors, p38 participates in transmission, amplification, and diversification of the extracellular signal, initiating several different cellular responses. Studies have revealed that activation of p38 pathway is related to many pathological changes that occur in the course of inflammatory/immunologic and cardiovascular diseases.

Gene engineering by selectable intraplasmid recombination: construction of novel dihydrofolate reductase minigenes.

An intraplasmid recombination system in Escherichia coli has been designed to make possible the engineering of various genes using methods that greatly reduce dependence on appropriately placed restriction enzyme sites. This system has been used to manipulate intervening sequences in dihydrofolate reductase minigenes and to vary the number of 48-bp repeats in the promoter region. In this method, the two fragments to be recombined are cloned into a plasmid separated by a fragment of DNA containing an expressible galactokinase-encoding gene (galK). Selection for loss of the galK gene, but for retention of the plasmid in E. coli, results in a plasmid in which the two fragments have undergone homologous recombination. Several new plasmids are reported here which contain an expressible galK gene flanked by multiple restriction sites. These plasmids should be useful in recombination and as convenient sources of a gene for which both positive and negative selections are available in E. coli.

Molecular cloning and characterization of a human protein kinase that specifically activates c-Jun N-terminal kinase.

The c-Jun N-terminal kinases (JNKs), also called stress-activated protein kinases (SAPKs), belong to the mitogen-activated protein kinase (MAPK) gene super-family. Like all the MAPKs, JNKs are activated through dual phosphorylation of a theronine residue and a tyrosine residue by a dual specificity kinase such as JNKK1/MKK4/SEK1. Here, we report the molecular cloning and characterization of hJNKK2 alpha, a human homolog of the recently reported murine MKK7 alpha. hJNKK2 alpha belongs to the MAPK kinase gene family and is expressed in many adult tissues. It is nearly identical to a recently reported human JNKK2 at the kinase domain but with major differences in both amino- and carboxyl-terminal sequences, suggesting that hJNKK2 alpha may be an alternative spliced form of this kinase. Expression of hJNKK2 alpha, but not its related kinases JNKK1/MKK4/SEK1, MEK1, MKK3, or MKK6, leads to strong activation of JNK in several cell lines. No activation of ERK or p38 kinases was observed with this kinase. An in-vitro kinase assay demonstrated that JNK1 activation by hJNKK2 alpha requires phosphorylation of the theronine and tyrosine residues at positions 183 and 185 in JNK1. Furthermore, hJNKK2 alpha activated the JNK-dependent signal transduction pathway in vivo by induction of c-Jun- and ATF2-mediated gene transcription. In conclusion, we have cloned the human homolog of murine MKK7 alpha, which may be an alternative spliced form of human JNKK2 involved in transducing specific upstream signals to regulate JNK activity in vivo.

Tgf-Beta promotes the growth of bovine chondrocytes in monolayer culture and the formation of cartilage tissue on three-dimensional scaffolds.

We have investigated the ability of transforming growth factor beta (TGF-beta) to promote the growth and differentiation of chondrocytes in monolayer and on three-dimensional scaffolds. Treatment of chondrocytes with TGF-beta and ascorbate individually stimulated the proliferation of bovine articular chondrocytes about 2-fold when cells were grown in monolayer culture: the combination of TGF-beta and ascorbate resulted in a 3-fold increase in cell number over a 72-h period. Peak stimulation with TGF-beta occurred at about 1.0 ng/ml: bFGF was slightly inhibitory in these assays. TGF-beta led to an increase in glycosaminoglycan synthesis as detected by Western blotting using anti-chondroitin sulfate antibodies. No significant change in collagen type II mRNA or protein was observed. When cells were grown on grown on three-dimensional scaffolds composed of polyglyocolic acid, TGF-beta treatment led to an increase in the size of the cartilage-like constructs produced. This was accompanied by increases in collagen and glycosaminoglycan deposition; immunohistochemical staining showed that the predominant collagen was type II. These results indicate that TGF-beta is capable of increasing the proliferation rate of chondrocytes in monolayer as well as increasing cartilage production on three-dimensional scaffolds and may find utility in the in vitro engineering of cartilage tissue.

Discrepant toxoplasma latex agglutination test results.

The analysis of 4450 toxoplasma serology results showed that 59 (1.3%) latex agglutination reactions were not confirmed in the dye test. These discrepant results were associated with an unspecified IgM antibody but not associated with kit batch variation, inactivation of sera, concurrent cytomegalovirus infection, or the presence of hepatitis B virus "e" antigen. The latex agglutination test is useful as a screen for toxoplasma infection but false positive reactions do occur. Patients at risk of severe toxoplasmosis should be investigated by additional tests.

Assessment of immunoglobulin-M immunosorbent agglutination assay (ISAGA) for detecting toxoplasma specific IgM.

An immunoglobulin-M immunosorbent agglutination assay (ISAGA) was introduced to detect toxoplasma specific IgM. This assay incorporates mu chain capture and use of entire toxoplasma trophozoites as an antigen source. The performance of the ISAGA was compared with that of a double sandwich enzyme linked immunosorbent assay (DS-ELISA) currently used in the Public Health Laboratory Service Toxoplasma Reference Laboratories. The ISAGA was found to be more sensitive than DS-ELISA but there was no demonstrable difference in the specificity or reproducibility between the two assays. The ISAGA is suitable for the diagnosis of acute toxoplasmosis in immunocompetent patients and as a screening test for recent infection in pregnant women. The persistence of ISAGA reactivity, however, is such that additional serological assessment is required to define the risk of congenital infection.

Direct agglutination test and other assays for measuring antibodies to Toxoplasma gondii.

The performance of a direct agglutination test for the detection of toxoplasma specific IgG immunoglobulin was compared with that of the latex agglutination test. The direct agglutination test was less sensitive but more specific than the latex agglutination test. Quantitative results were not directly comparable, reflecting the different antibody profiles detected in each assay. The direct agglutination test represents an alternative to the latex agglutination test as a screening test for toxoplasmosis. Patients at risk of life threatening infection require detailed serological examination using additional assays.

Comparison of two methods for enumerating malaria parasites in thick blood films.

Calculation of parasite densities is important for estimating herd immunity to malaria, and for determining end points in field trials for interventions such as malaria vaccines, impregnated bed nets, and chemosuppression. Two methods of enumeration were compared: method 1, in which 100 consecutive high-power fields (HPFs) are examined, and if they all contain at least one parasite, the number per field is then counted in 10-100 of these fields according to density; and method 2, in which the actual number of parasites present in 100 consecutive fields are counted. The first method significantly underestimates parasite density in samples in which less than all high-power fields are parasite-positive. A correction of method 1 is suggested, which results in a parasite density, which is comparable with that obtained using method 2. The correction factor estimated was 2(-In(1 - p)), where p is the proportion of positive HPFs. The correction factor presented will allow accurate estimate of parasite densities per volume of blood even if only the proportion of parasite-positive high-power fields containing at least one parasite are counted.

Acridine orange fluorescence techniques as alternatives to traditional Giemsa staining for the diagnosis of malaria in developing countries.

Traditional Giemsa-stained thick blood films were compared with 2 fluorescence microscopy techniques, acridine orange (AO) staining of thin blood films and the quantitative buffy coat (QBC) method, for the microscopical diagnosis of malaria. Of 200 samples examined, 141 were positive by Giemsa staining, 146 by AO and 137 by QBC. Overall sensitivities for the 2 fluorescence techniques compared to Giemsa staining were good: AO 97.9% and QBC 93.6%. However, with parasitaemias < 100/microL the QBC sensitivity fell to 41.7% whereas that of AO was 83.3%. Both AO and QBC were unable to differentiate accurately between individual malaria species. We conclude that the QBC technique alone cannot replace Giemsa-stained thick blood films for most purposes in an African setting. However, apart from species differentiation, the AO method is an appropriate technique for the laboratory diagnosis of malaria in developing countries.

Epileptic seizures and malaria in Kenyan children.

Between October 1990 and November 1991 data were collected on the frequency, causes, and nature of epileptic seizures in children admitted to the paediatric ward at Kilifi District Hospital, Kenya, from a defined study area. During this period, 1324 children were studied, of whom 15.8% had seizures as part of their illness. Malaria was by far the commonest cause of seizures, accounting for 69.0%; no other single condition caused more than 4.4%. The proportion of respiratory infections complicated by seizures was 4.0% compared to 31.3% for malaria. Only 25% of malaria-related epileptic seizures were associated with cerebral malaria; the remainder were associated with otherwise uncomplicated malaria and, in this group, 84% had complex seizures, with 47% being partial and over 70% repetitive. There was no relationship with fever, with 54% of observed seizures occurring at rectal temperatures below 38 degrees C. The minimum community incidence of complex seizures in association with non-cerebral malaria was 5.8 per 1000 per year. Complex epileptic seizures in association with otherwise uncomplicated malaria are common and may be a significant cause of longer term morbidity in malaria endemic areas.

Malaria is an important cause of anaemia in primigravidae: evidence from a district hospital in coastal Kenya.

A study was undertaken in order to determine the prevalence and aetiology of anaemia in pregnancy in coastal Kenya, so as to establish locally important causes and enable the development of appropriate intervention strategies. 275 women attending the antenatal clinic at Kilifi district hospital, Kenya, were recruited in November 1993. The prevalence of anaemia (haemoglobin [Hb] < 11 g/dL) was 75.6%, and the prevalence of severe anaemia (Hb < 7g/dL) was 9.8% among all parities; 15.3% of 73 primigravidae were severely anaemic, compared with 7.9% of 202 multigravidae (P = 0.07). In primigravidae, malaria infection (Plasmodium falciparum) was strongly associated with moderate and severe anaemia (chi 2 test for trend, P = 0.003). Severe anaemia was more than twice as common in women with peripheral parasitaemia as in those who were aparasitaemic, and parasitaemia was associated with a 2.2g/dL decrease in mean haemoglobin level (P < 0.001). In multigravidae, iron deficiency and hookworm infection were the dominant risk factors for anaemia. Folate deficiency and human immunodeficiency virus infection were not strongly associated with anaemia. It is suggested that an intervention that can effectively reduce malaria infection in primigravidae could have a major impact on the health of these women and their infants.

The Gambian National Impregnated Bednet Programme: costs, consequences and net cost-effectiveness.

Clinical trials have indicated that treating mosquito nets with insecticide could be a potentially cost-effective method of preventing malaria. As malaria is one of the most common causes of death in children under five in developing countries, there has been substantial interest in whether such findings can be replicated for a country's control programme in practice. The cost-effectiveness of the Gambian National Insecticide-impregnated Bednet Programme (NIBP), from the viewpoint of providers (government and non-governmental agencies) and the community, has been calculated. Information was collected from existing records, interviews with NIBP personnel, observation and household surveys. Information is provided on the resource use consequences of the NIBP in terms of reduced expenditure on anti-malaria preventive measures, treatment in government health services, household financed treatment and "charity" (burial, funeral and mourning activities), as well as cash income lost as a result of child death. The annual implementation cost of the NIBP was D757,875 (US$91,864), of which 86% was recurrent cost. The estimated number of death averted was 40.56. The net implementation cost-effectiveness ratio per death averted and discounted life years gained were D3884 (US$471) and D260 (US$31.5), respectively. Adding the cost of all mosquito nets would increase the cost-effectiveness ratios by over five times, which is an important consideration for countries with a lower coverage of mosquito nets per capita. It is concluded that insecticide-impregnated mosquito nets are one of the more efficient ways of reducing deaths in children under 10 years in rural Gambia.

Bronchoalveolar lavage in the prediction of post-thoracotomy chest infection.

A prospective study was carried out on 75 patients undergoing pulmonary surgery to determine the relationship between perioperative lung flora and postoperative infections. Seventy-five patients having pleurectomy or pneumonectomy received cefuroxime prophylaxis; 1.5 g i.v. at induction followed by 6 further doses of 0.75 g i.v. over 48 h. Bronchoalveolar lavage samples were taken perioperatively via bronchoscopy in pleurectomy patients and from excised lung in patients undergoing lung resection. Patients were monitored for development of chest infection during the first 10 days after operation. Bacterial pathogens were cultured from 12 out of 54 lavage samples. The most common pathogen was Haemophilus influenzae and all the organisms were sensitive to cefuroxime. Eight patients (10.7%) developed postoperative chest infection. The likelihood of developing postoperative chest infection was 42% (5 out of 12 patients) in those patients whose lavage culture was positive for bacterial pathogens compared to 4.8% (2 out of 42 patients) for those whose culture was negative (chi 2 test, p less than 0.001). These results suggest that the culturing of bacterial pathogens from lavage samples from resected lung is a significant predictor of postoperative chest infection in patients undergoing pulmonary resection.

The links between health and the political structure in New China.

PIP: Data collected on barefoot doctors by the authors in the course of a 5-week visit to China in 1973 is presented, and the recent recommendation, made by others, that the concept of barefoot doctors should be transplanted to Western countries is criticized. Barefoot doctors in China function within an educational, medical, political, and social framework which differs greatly from that found in Western countries. In countries, such as the United States, barefoot doctors could be trained easily, but they could not be utilized effectively without instituting extensive changes in the social structure of these countries. In China basic health directives are issued by the central government, but the manner in which these directives are implemented is determined by the members of each community. In the U.S., participation and involvement in decision making on the part of community members is minimal and most health decisions are made by the dominant elite. In China the majority of the barefoot doctors are recruited for training from rural areas and most of them return to their own communs after they have completed their training. Training time varies and depends on the specific medical needs of the community where the barefoot doctor will serve. Barefoot doctors generally devote 1/3 of their time to medical practice and the other 2/3 of their time is spent working in the fields. Community residents pay the salary of the barefoot doctors and many communes have their own health insurance plans. Barefoot doctors play an important role in the delivery of family planning services. They pass out oral contraceptives, give injectables, insert IUDs, and perform vasectomies. Most communes keep records on the number of children born to each woman and on the contraceptive methods used by each couple. Family planning is a joint undertaking of the entire community. By analyzing the role of the barefoot doctor, the authors were able to identify fundamental differences in the health care system of China and the U.S.^ieng

Distribution study of orally administered lipoic acid in rat brain tissues.

Lipoic acid (LA), an essential cofactor for mitochondrial enzymes and a natural antioxidant, has been explored for the treatment of Alzheimer's disease. However, lipoic acid distribution in brain has not been investigated via oral dosing in human subjects or animals. Therefore, we aim to investigate the distribution of orally administered LA from systemic circulation into rat brain tissues and understand the transport efficiency of lipoic acid across the blood-brain barrier. Brain and blood samples were obtained from male Lister Hooded rats at pre-defined time points after single and chronic oral dosing of LA at 50 mg/kg. Levels of LA were determined using liquid chromatography tandem mass spectrometry. An equilibrium dialysis method was employed to elucidate LA protein binding in brain and blood tissues. Basal endogenous levels of LA in control rats were found to fluctuate between 0.005 and 0.267 microM in blood and 0-0.024 microM in brain after correction for residual blood volume. Pharmacokinetic profiling demonstrated rapid biphasic elimination of LA in blood and poor distribution into various brain regions with levels ranging from 0.0009 to 0.0072 microM. The in vitro and in vivo LA brain:blood partition ratios were 0.1 and -0.01, respectively. Our results demonstrate for the first time that LA does not cross the blood-brain barrier readily and suggest that the antioxidant effect of LA in brain may not be due to its direct effect in the central nervous system.