Validation and adaptation of rapid neurodevelopmental assessment instrument for infants in Guatemala.

BACKGROUND: Timely detection of neurodevelopmental impairments in children can prompt referral for critical services that may prevent permanent disability. However, screening of impairments is a significant challenge in low-resource countries. We adapted and validated the rapid neurodevelopmental assessment (RNDA) instrument developed in Bangladesh to assess impairment in nine domains: primitive reflexes, gross and fine motor development, vision, hearing, speech, cognition, behaviour and seizures. METHODS: We conducted a cross-sectional study of 77 infants (0-12 months) in rural Guatemala in July 2012 and July 2013. We assessed inter-rater reliability and predictive validity between the 27-item RNDA and the 325-item Bayley Scales of Infant and Toddler Development, Third Edition (BSID-III) and concurrent validity based on chronic malnutrition, a condition associated with neurodevelopmental impairments. For both RNDA and BSID-III, standardized scores below 80 were defined as borderline impairment. RESULTS: Children came from rural households (92%), were born to indigenous women of Mayan descent (73%) and had moderate or severe growth stunting (43%). Inter-rater reliability for eight RNDA domains was of moderate to high reliability (weighted κ coefficients, 0.49-0.99). Children screened positive for impairment in fine motor (17%) and gross motor (14%) domains using the RNDA. The RNDA had good concurrent ability; infants who were growth stunted had higher mean levels of impairment in gross motor, speech and cognition domains (all p < 0.001). The RNDA took 20-30 min to complete compared with 45-60 min for BSID-III. CONCLUSIONS: Wide-scale implementation of a simple, valid and reliable screening tool like the RNDA by community health workers would facilitate early screening and referral of infants at-risk for neurodevelopmental impairment.

Knee angle-dependent oxygen consumption of human quadriceps muscles during maximal voluntary and electrically evoked contractions.

Fatigability and muscle oxygen consumption (mVO(2)) during sustained voluntary isometric knee extensions are less at extended (30 degrees knee angle; 0 degrees , full extension) versus flexed knee angles (90 degrees). This lower energy consumption may partially result from lower neural activation at extended knee angles. We hypothesized a smaller difference in mVO(2) between extended and flexed knee angles during electrical stimulation, which guaranteed maximal activation, than during maximal voluntary contractions (MVC). In eight healthy young males, MVC extension torque was obtained at 30 degrees, 60 degrees and 90 degrees knee angles. mVO(2) of the rectus femoris (RF), vastus lateralis (VL) and medialis muscle was measured using near-infrared spectroscopy during tetanic (10 s) and maximal voluntary (15 s) contractions (MVC(15)). For electrically induced contractions, steady state mVO(2) was reached at similar (P > 0.05) times after torque onset (4.6 +/- 0.7 s) at all knee angles. In contrast, during MVC(15) at 30 degrees mVO(2) was reached at 7.1 +/- 1.1 s, significantly later compared to 60 degrees and 90 degrees knee angles. The knee angle dependent differences in mVO(2) were not lower in electrically induced contractions (as hypothesised) but were similar as in voluntary contractions. Normalized mVO(2) at 30 degrees (percentage 90 degrees knee angle) was 79.0 +/- 9.4% (across muscles) for electrically induced and 79.5 +/- 7.6% (across muscles) for voluntary contractions (P < 0.05). We conclude that the slower onset of mVO(2) during voluntary effort at 30 degrees may have been due to a lower maximal activation. However, because steady state mVO(2) both during electrically induced and voluntary contractions was approximately 20% less at extended versus flexed knee angles, the causes for the lower mVO(2) must reside within the muscle itself.

Conventionally assessed voluntary activation does not represent relative voluntary torque production.

The ability to voluntarily activate a muscle is commonly assessed by some variant of the twitch interpolation technique (ITT), which assumes that the stimulated force increment decreases linearly as voluntary force increases. In the present study, subjects (n = 7) with exceptional ability for maximal voluntary activation (VA) of the knee extensors were used to study the relationship between superimposed and voluntary torque. This includes very high contraction intensities (90-100%VA), which are difficult to consistently obtain in regular healthy subjects (VA of approximately 90%). Subjects were tested at 30, 60, and 90 degrees knee angles on two experimental days. At each angle, isometric knee extensions were performed with supramaximal superimposed nerve stimulation (triplet: three pulses at 300 Hz). Surface EMG signals were obtained from rectus femoris, vastus lateralis, and medialis muscles. Maximal VA was similar and very high across knee angles: 97 +/- 2.3% (mean +/- SD). At high contraction intensities, the increase in voluntary torque was far greater than would be expected based on the decrement of superimposed torque. When voluntary torque increased from 79.6 +/- 6.1 to 100%MVC, superimposed torque decreased from 8.5 +/- 2.6 to 2.8 +/- 2.3% of resting triplet. Therefore, an increase in VA of 5.7% (from 91.5 +/- 2.6 to 97 +/- 2.3%) coincided with a much larger increase in voluntary torque (20.4 +/- 6.1%MVC) and EMG (33.9 +/- 6.6%max). Moreover, a conventionally assessed VA of 91.5 +/- 2.6% represented a voluntary torque of only 79.6 +/- 6.1%MVC. In conclusion, when maximal VA is calculated to be approximately 90% (as in regular healthy subjects), this probably represents a considerable overestimation of the subjects' ability to maximally drive their quadriceps muscles.

Knee extensor muscle oxygen consumption in relation to muscle activation.

Recently, fatigability and muscle oxygen consumption (mVO(2)) during sustained isometric contractions were found to be less at shorter (30 degrees knee angle; 0 degrees = full extension) compared to longer knee extensor muscle lengths (90 degrees ) and, at low torques, less in the rectus femoris (RF) muscle than in the vastus lateralis and medialis. In the present study we hypothesized that these findings could be accounted for by a knee angle- and a muscle-dependent activation respectively. On two experimental days rectified surface EMG (rsEMG) was obtained as a measure of muscle activation in nine healthy young males. In addition, on day 1 maximal torque capacity (MTC) was carefully determined using superimposed nerve stimulation on brief high intensity contractions (> 70%MVC) at 30, 60 and 90 degrees knee angles. On day 2, subjects performed longer lasting isometric contractions (10-70%MTC) while mVO(2) was measured using near-infrared spectroscopy (NIRS). At 30 degrees , maximal mVO(2) was reached significantly later (11.0 s +/- 6.5 s) and was 57.9 +/- 8.3% less (average +/- SD, across intensities and muscles) than mVO(2) at 60 and 90 degrees (p < 0.05). However, rsEMG was on average only 18.0 +/- 11.8% (p = 0.062) less at the start of the contraction at 30 degrees . At 10%MTC at all knee angles, maximal mVO(2) of the RF occurred significantly later (28.8 +/- 36.0 s) and showed a significantly smaller increase in rsEMG compared to both vasti. In conclusion, it is unlikely that the tendency for less intense muscle activation could fully account for the approximately 60% lower oxygen consumption at 30 degrees , but the later increase in RFmVO(2) seemed to be caused by a less strong activation of the RF.

Muscle activation and blood flow do not explain the muscle length-dependent variation in quadriceps isometric endurance.

We investigated the role of central activation in muscle length-dependent endurance. Central activation ratio (CAR) and rectified surface electromyogram (EMG) were studied during fatigue of isometric contractions of the knee extensors at 30 and 90 degrees knee angles (full extension = 0 degree). Subjects (n = 8) were tested on a custom-built ergometer. Maximal voluntary isometric knee extension with supramaximal superimposed burst stimulation (three 100-mus pulses; 300 Hz) was performed to assess CAR and maximal torque capacity (MTC). Surface EMG signals were obtained from vastus lateralis and rectus femoris muscles. At each angle, intermittent (15 s on 6 s off) isometric exercise at 50% MTC with superimposed stimulation was performed to exhaustion. During the fatigue task, a sphygmomanometer cuff around the upper thigh ensured full occlusion (400 mmHg) of the blood supply to the knee extensors. At least 2 days separated fatigue tests. MTC was not different between knee angles (30 degrees : 229.6 +/- 39.3 N.m vs. 90 degrees: 215.7 +/- 13.2 N.m). Endurance times, however, were significantly longer (P < 0.05) at 30 vs. 90 degrees (87.8 +/- 18.7 vs. 54.9 +/- 12.1 s, respectively) despite the CAR not differing between angles at torque failure (30 degrees: 0.95 +/- 0.05 vs. 90 degrees: 0.96 +/- 0.03) and full occlusion of blood supply to the knee extensors. Furthermore, rectified surface EMG values of the vastus lateralis (normalized to prefatigue maximum) were also similar at torque failure (30 degrees : 56.5 +/- 12.5% vs. 90 degrees : 58.3 +/- 15.2%), whereas rectus femoris EMG activity was lower at 30 degrees (44.3 +/- 12.4%) vs. 90 degrees (69.5 +/- 25.3%). We conclude that differences in endurance at different knee angles do not find their origin in differences in central activation and blood flow but may be a consequence of muscle length-related differences in metabolic cost.

Initial phase of maximal voluntary and electrically stimulated knee extension torque development at different knee angles.

We investigated the capacity for torque development and muscle activation at the onset of fast voluntary isometric knee extensions at 30, 60, and 90 degrees knee angle. Experiments were performed in subjects (n = 7) who had high levels (>90%) of activation at the plateau of maximal voluntary contractions. During maximal electrical nerve stimulation (8 pulses at 300 Hz), the maximal rate of torque development (MRTD) and torque time integral over the first 40 ms (TTI40) changed in proportion with torque at the different knee angles (highest values at 60 degrees ). At each knee angle, voluntary MRTD and stimulated MRTD were similar (P < 0.05), but time to voluntary MRTD was significantly longer. Voluntary TTI40 was independent (P > 0.05) of knee angle and on average (all subjects and angles) only 40% of stimulated TTI40. However, among subjects, the averaged (across knee angles) values ranged from 10.3 +/- 3.1 to 83.3 +/- 3.2% and were positively related (r2 = 0.75, P < 0.05) to the knee-extensor surface EMG at the start of torque development. It was concluded that, although all subjects had high levels of voluntary activation at the plateau of maximal voluntary contraction, among subjects and independent of knee angle, the capacity for fast muscle activation varied substantially. Moreover, in all subjects, torque developed considerably faster during maximal electrical stimulation than during maximal voluntary effort. At different knee angles, stimulated MRTD and TTI40 changed in proportion with stimulated torque, but voluntary MRTD and TTI40 changed less than maximal voluntary torque.

The Drosophila melanogaster DmRAD54 gene plays a crucial role in double-strand break repair after P-element excision and acts synergistically with Ku70 in the repair of X-ray damage.

The RAD54 gene has an essential role in the repair of double-strand breaks (DSBs) via homologous recombination in yeast as well as in higher eukaryotes. A Drosophila melanogaster strain deficient in the RAD54 homolog DmRAD54 is characterized by increased X-ray and methyl methanesulfonate (MMS) sensitivity. In addition, DmRAD54 is involved in the repair of DNA interstrand cross-links, as is shown here. However, whereas X-ray-induced loss-of-heterozygosity (LOH) events were completely absent in DmRAD54(-/-) flies, treatment with cross-linking agents or MMS resulted in only a slight reduction in LOH events in comparison with those in wild-type flies. To investigate the relative contributions of recombinational repair and nonhomologous end joining in DSB repair, a DmRad54(-/-)/DmKu70(-/-) double mutant was generated. Compared with both single mutants, a strong synergistic increase in X-ray sensitivity was observed in the double mutant. No similar increase in sensitivity was seen after treatment with MMS. Apparently, the two DSB repair pathways overlap much less in the repair of MMS-induced lesions than in that of X-ray-induced lesions. Excision of P transposable elements in Drosophila involves the formation of site-specific DSBs. In the absence of the DmRAD54 gene product, no male flies could be recovered after the excision of a single P element and the survival of females was reduced to 10% compared to that of wild-type flies. P-element excision involves the formation of two DSBs which have identical 3' overhangs of 17 nucleotides. The crucial role of homologous recombination in the repair of these DSBs may be related to the very specific nature of the breaks.

Identification and characterisation of the Drosophila melanogaster O6-alkylguanine-DNA alkyltransferase cDNA.

The protein O 6-alkylguanine-DNA alkyltransferase(alkyltransferase) is involved in the repair of O 6-alkylguanine and O 4-alkylthymine in DNA and plays an important role in most organisms in attenuating the cytotoxic and mutagenic effects of certain classes of alkylating agents. A genomic clone encompassing the Drosophila melanogaster alkyltransferase gene ( DmAGT ) was identified on the basis of sequence homology with corresponding genes in Saccharomyces cerevisiae and man. The DmAGT gene is located at position 84A on the third chromosome. The nucleotide sequence of DmAGT cDNA revealed an open reading frame encoding 194 amino acids. The MNNG-hypersensitive phenotype of alkyltransferase-deficient bacteria was rescued by expression of the DmAGT cDNA. Furthermore, alkyltransferase activity was identified in crude extracts of Escherichia coli harbouring DmAGT cDNA and this activity was inhibited by preincubation of the extract with an oligonucleotide containing a single O6-methylguanine lesion. Similar to E.coli Ogt and yeast alkyltransferase but in contrast to the human alkyltransferase, the Drosophila alkyltransferase is resistant to inactivation by O 6-benzylguanine. In an E.coli lac Z reversion assay, expression of DmAGT efficiently suppressed MNNG-induced G:C-->A:T as well as A:T-->G:C transition mutations in vivo. These results demonstrate the presence of an alkyltransferase specific for the repair of O 6-methylguanine and O 4-methylthymine in Drosophila.

The Drosophila melanogaster RAD54 homolog, DmRAD54, is involved in the repair of radiation damage and recombination.

The RAD54 gene of Saccharomyces cerevisiae plays a crucial role in recombinational repair of double-strand breaks in DNA. Here the isolation and functional characterization of the RAD54 homolog of the fruit fly Drosophila melanogaster, DmRAD54, are described. The putative Dmrad54 protein displays 46 to 57% identity to its homologs from yeast and mammals. DmRAD54 RNA was detected at all stages of fly development, but an increased level was observed in early embryos and ovarian tissue. To determine the function of DmRAD54, a null mutant was isolated by random mutagenesis. DmRADS4-deficient flies develop normally, but the females are sterile. Early development appears normal, but the eggs do not hatch, indicating an essential role for DmRAD54 in development. The larvae of mutant flies are highly sensitive to X rays and methyl methanesulfonate. Moreover, this mutant is defective in X-ray-induced mitotic recombination as measured by a somatic mutation and recombination test. These phenotypes are consistent with a defect in the repair of double-strand breaks and imply that the RAD54 gene is crucial in repair and recombination in a multicellular organism. The results also indicate that the recombinational repair pathway is functionally conserved in evolution.

Two ras genes in Dictyostelium minutum show high sequence homology, but different developmental regulation from Dictyostelium discoideum rasD and rasG genes.

The social amoeba Dictyostelium discoideum expresses five ras genes at different stages of development. One of them, DdrasD is expressed during postaggregative development and transcription is induced by extracellular cAMP. A homologue of DdrasD, the DdrasG gene, is expressed exclusively during vegetative growth. We cloned two ras homologues Dmras1 and Dmras2 from the primitive species D. minutum, which show high homology to DdrasD and DdrasG and less homology to the other Ddras genes. In contrast to the DdrasD and DdrasG genes, both the Dmras1 and Dmras2 genes are expressed during the entire course of development. The expression levels are low during growth, increase at the onset of starvation and do not decrease until fruiting bodies have formed. Expression of neither Dmras1 or Dmras2 is regulated by cAMP. So even though the high degree of homology between the ras genes of different species suggests conservation of function, this function is apparently not associated with a specific developmental stage.