Impact of expression mode and timing of sample collection, relative to milk ejection, on human milk bacterial DNA profiles.

AIM: To investigate the impact of expression mode: electric breast pump or hand expression, and timing of sample collection: pre- and post-milk ejection on human milk (HM) bacterial DNA profiles. METHODS AND RESULTS: Three HM samples from the same breast were collected from 30 breastfeeding mothers: a pre-milk ejection pump-expressed sample (pre-pump), a post-milk ejection pump-expressed sample (post-pump) and a post-milk ejection hand-expressed sample (post-hand). Full-length 16S rRNA gene sequencing was used to assess milk bacterial DNA profiles. Bacterial profiles did not differ significantly based on mode of expression nor timing of sample collection. No significant differences were detected in the relative abundance of any OTUs based on expression condition (pre-pump/ post-pump and post-pump/post-hand) with univariate linear mixed-effects regression analyses (all P-values > 0·01; α = 0·01). Similarly, no difference in richness was observed between sample types (number of observed OTUs: post-pump/post-hand P = 0·13; pre-pump/post-pump P = 0. 45). CONCLUSION: Bacterial DNA profiles of HM did not differ according to either expression method or timing of sample collection. SIGNIFICANCE AND IMPACT OF THE STUDY: Hand or pump expression can be utilized to collect samples for microbiome studies. This has implications for the design of future HM microbiome studies.

DNA extraction method influences human milk bacterial profiles.

AIMS: To evaluate four DNA extraction methods to elucidate the most effective method for bacterial DNA recovery from human milk (HM). METHODS AND RESULTS: Human milk DNA was extracted using the following methods: (i) Qiagen MagAttract Microbial DNA Isolation Kit (kit QM), (ii) Norgen Milk Bacterial DNA Isolation Kit (kit NM), (iii) Qiagen MagAttract Microbiome DNA/RNA Isolation Kit (kit MM) and (iv) TRIzol LS Reagent (method LS). The full-length 16S rRNA gene was sequenced. Kits MM and method LS were unable to extract detectable levels of DNA in 9/11 samples. Detectable levels of DNA were recovered from all samples using kits NM (mean = 0·68 ng µl-1 ) and QM (mean = 0·55 ng µl-1 ). For kits NM and QM, the greatest number of reads were associated with Staphylococcus epidermidis, Streptococcus vestibularis, Propionibacterium acnes, Veillonella dispar and Rothia mucilaginosa. Contamination profiles varied substantially between kits, with one bacterial species detected in negative extraction controls generated with kit QM and six with kit NM. CONCLUSIONS: Kit QM is the most suitable of the kits tested for the extraction of bacterial DNA from human milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Choice of extraction method impacts the efficiency of bacterial DNA extraction from human milk and the resultant bacterial community profiles generated from these samples.

Re: "Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community" (2018) 6:87, https://doi.org/10.1186/s40168-018-0475-7.

A recent publication by Lim et al. 2018 (Amniotic fluid from healthy term pregnancies does not harbor a detectable microbial community) strongly concluded that the microbiome of amniotic fluid primarily originates from reagent contamination. However, upon closer inspection of the methods used and data presented in this study, in particular the supplementary data, such conclusions do not appear to be supported by the results. We outline such methodological/data interpretation concerns and invite the authors to discuss these.

Characterization of the bacterial microbiome in first-pass meconium using propidium monoazide (PMA) to exclude nonviable bacterial DNA.

Numerous studies have reported bacterial DNA in first-pass meconium samples, suggesting that the human gut microbiome is seeded prior to birth. However, these studies have not been able to discriminate between DNA from living bacterial cells, DNA from dead bacterial cells or cell-free DNA. Here we have used propidium monoazide (PMA) together with 16S rRNA gene sequencing to determine whether there are intact bacterial cells in the fetal gut. DNA was extracted from first-pass meconium (n = 5) and subjected to 16S rRNA gene sequencing with/without PMA treatment. All meconium samples, regardless of PMA treatment, contained detectable levels of bacterial DNA; however, treatment with PMA prior to DNA extraction decreased the DNA yield by approximately 20%. PMA-treated meconium samples did not differ significantly from untreated samples in terms of observed number of OTUs (P = 0·945); although they did differ taxonomically, with around one quarter of OTUs identified in untreated samples only, suggesting that they have originated from cell-free/nonviable DNA. The mean Sørensen coefficient for treated vs untreated samples was 0·527. Our findings suggest that the fetal gut is seeded with intact bacterial cells prior to birth. This is an important finding, as exposure to live bacteria during gestation might have a significant impact on the developing fetus. SIGNIFICANCE AND IMPACT OF THE STUDY: DNA-based microbiome studies performed using 16S rRNA gene sequencing are limited by their inability to discriminate between live bacterial cells, dead bacterial cells and cell-free DNA. Here we use propidium monoazide (PMA) to exclude nonviable bacteria from microbiome analysis of first-pass meconium samples and thereby reveal that the majority of the purported fetal gut microbiome is from intact bacterial cells. This work demonstrates the importance of excluding nonviable bacteria when analysing the microbial community in low-biomass samples such as meconium.

A cost-utility analysis of decompressive hemicraniectomy versus medical treatment in the management of space-occupying brain oedema post middle cerebral artery infarction.

BACKGROUND AND PURPOSE: Data from randomly controlled trials have indicated that a decompressive hemicraniectomy is more clinically effective than medical treatment in the management of space-occupying brain oedema post middle cerebral artery infarction. This economic evaluation compares the impact of the two options in the UK. No recent study has conducted an economic evaluation on this topic for the UK. METHOD: A cost-utility analysis over a time period of 1 year was used, measuring benefits in terms of quality adjusted life years (QALYs) and costs in pound sterling, discounted to 2015 prices. The evaluation was from the perspective of the National Health Service, the largest healthcare provider in the UK. RESULTS: The cost-utility analysis found an incremental cost effectiveness of £116 595.10 for every QALY gained if patients were offered a decompressive hemicraniectomy compared to the best medical treatment. DISCUSSION: This is above the National Institute for Health and Care Excellence (NICE) 'cost-effective' threshold of £20 000-£30 000 per QALY, but lower mortality rates associated with the surgical alternative raises ethical considerations for healthcare providers in the UK.

Identification and removal of contaminating microbial DNA from PCR reagents: impact on low-biomass microbiome analyses.

Reagent-derived contamination can compromise the integrity of microbiome data, particularly in low microbial biomass samples. This contamination has recently been attributed to the 'kitome' (contamination introduced by the DNA extraction kit), prior to which attention was mostly paid to potential contamination introduced by PCR reagents. In this study, we assessed the proportion to which our DNA extraction kit and PCR master mix introduce contaminating microbial DNA to bacterial microbial profiles generated by 16S rRNA gene sequencing. Utilizing a commercial dsDNase treatment protocol to decontaminate the PCR master mix, we demonstrated that the vast majority of contaminating DNA was derived from the PCR master mix. Importantly, this contamination was almost completely eliminated using the simple dsDNase treatment, resulting in a 99% reduction in contaminating bacterial reads. We suggest that dsDNase treatment of PCR reagents should be explored as a simple and effective way of reducing contamination in low-biomass microbiome studies and producing more robust and reliable data. SIGNIFICANCE AND IMPACT OF THE STUDY: Reagent contamination with microbial DNA is a major problem in microbiome studies of low microbial biomass samples. Levels of such contaminating DNA often outweigh what is present in the sample and heavily confound subsequent data analysis. Previous studies have suggested this contamination is primarily derived from DNA extraction kits. Here, we identified the PCR master mix as the primary source of contamination, and showed that enzymatic removal of the contamination drastically reduced the blank signal and improved precision. Decontamination of PCR master mixes may have the potential to improve the sensitivity and accuracy of low-biomass microbiome studies.

Peripheral Subcutaneous Electrostimulation for Control of Intractable Post-operative Inguinal Pain: A Case Report Series.

Objective. To present a novel treatment approach for intractable inguinal neuralgia utilizing percutaneous peripheral electrostimulation. Materials and Methods. We report results of peripheral electrostimulation in three patients who experienced onset of inguinal neuralgic pain in the immediate postoperative period following herniorrhaphy. In each case pain was refractory to medical management and caused significant functional impairment. These three patients were first percutaneously trialed and then implanted with dual octipolar peripheral nerve stimulators (Advanced Neuromodulation Systems, Plano, TX) in close proximity to the injured nerve. Results. All three patients responded very favorably (75-100% pain relief at three, 10, and 12 months postimplantation). There were no complications. All three tapered off their pain-related medication, dramatically increased their levels of activity, and discontinued pain-related medical visits. Both patients who were on disability due to inguinal pain have been able to return to full-time employment. Conclusion. These preliminary results suggest that the technique described is effective and safe, and may be cost-effective as well.

Characteristics of clients with schizophrenia who express certainty or uncertainty about continuing treatment with depot neuroleptic medication.

In this exploratory study, the factors corresponding to clients' certainty or uncertainty in continuing with depot neuroleptic medication for schizophrenia were examined. Ninety-four participants from a tertiary care schizophrenia clinic received an educational intervention aid containing information about treatment risks and benefits and were interviewed to elicit their levels of decisional conflict, self-efficacy, and emotional support, as well as expectations of risks and benefits of treatment. Eighty-seven percent of participants decided to continue treatment. Clients who expressed uncertainty (10%) about continuing with treatment had higher levels of decisional conflict (p = .000), lower levels of decision self-efficacy (p = .037) and decision emotional control (p = .003), lower expectations of hospitalization if treatment was stopped (p = .04) as well as lower expectations of benefits and higher expectations of side effects (p = .04), if treatment continued. In addition, reasons identified by participants for certainty or uncertainty were reported. The research may be useful in increasing the awareness of clinicians about the factors contributing to treatment decision making by clients diagnosed with schizophrenia and ultimately improve collaborative decision making.

Evaluation of cisatracurium, a new neuromuscular blocking agent, for tracheal intubation.

PURPOSE: The primary objective of this study was a blinded, randomized comparison of the recommended intubating dose of atracurium (0.5 mg. kg-1) with an approximately equipotent dose of cisatracurium (0.1 mg. kg-1) during N2O/O2/propofol/fentanyl anaesthesia. METHODS: Eighty ASA physical status 1 or 2 patients, 18-70 yr of age, within 30% of ideal body weight, scheduled for elective low to moderate risk surgical procedures were studied. Adductor pollicis evoked twitch responses were measured with a Grass FT 10 force displacement transducer (Grass Instruments, Quincy, MA) and continuously recorded on a Gould multichannel polygraph (Gould Instrument Systems, Cleveland, OH) after induction of anaesthesia. RESULTS: Increasing the initial dose of cisatracurium (from 0.1 to 0.15 and 0.2 mg. k-1, decreased mean time of onset (from 4.6 to 3.4 and 2.8 min, respectively), and increased mean time of clinically effective duration (45 to 55 and 61 min, respectively). Recovery to a T4:T1 ratio of 0.7 occurred approximately seven minutes following administration of the reversal agent neostigmine for all treatment groups. Intubation conditions were good or excellent in over 90% of patients in all treatment groups (two minutes after approximately 2 x ED95 doses of cisatracurium or atracurium and 1.5 minutes after 3 x and 4 x ED95 doses of cisatracurium). CONCLUSION: The intubation results reported in this study together with the combination of predictable recovery from neuromuscular block and apparent haemodynamic stability make cisatracurium a potentially useful muscle relaxant in clinical practice.

Train-of-four recovery after pharmacologic antagonism of pancuronium-, pipecuronium-, and doxacurium-induced neuromuscular block in anaesthetized humans.

Previous studies have suggested that the increased duration of action of long-acting neuromuscular relaxants may make their pharmacologic antagonism more difficult and, thus, increase the likelihood of residual block. This hypothesis was tested in healthy, adult humans who received a background of isoflurane/N2O/fentanyl anaesthesia. Study subjects were paralyzed with either pancuronium (N = 8), pipecuronium (N = 8), or the longer-acting relaxant, doxacurium (N = 8). Neuromuscular function was monitored, and, using a blinded, randomized study design, the relaxants were titrated to identify the ED95 dose in each patient. Thereafter, spontaneous recovery was observed until there was 25% of baseline response to the first supramaximal twitch (T1) in a train-of-four (TOF). At this time, the block was antagonized with neostigmine 0.07 mg/kg and glycopyrrolate 0.014 mg/kg i.v., and recovery of TOF was recorded. Spontaneous recovery to 25% of the baseline T1 response occurred at 52 +/- 14 min (mean +/- SD) following administration of either pancuronium and pipecuronium, and 85 +/- 33 min following doxacurium (P < 0.05 for doxacurium versus pancuronium and pipecuronium). In doxacurium-treated patients, reversal of block with neostigmine was less predictable and less complete than with the other two relaxants. For example, the ratio of the fourth to first twitch (T4/T1) of the TOF at 10 and 15 min after reversal was significantly less with doxacurium (59 +/- 14% and 61 +/- 16%, respectively) than with either pancuronium (75 +/- 6% and 75 +/- 10%) or pipecuronium (76 +/- 9% for both).(ABSTRACT TRUNCATED AT 250 WORDS)

A computer-controlled, closed-loop infusion system for infusing muscle relaxants: its use during motor-evoked potential monitoring.

A microcomputer-controlled closed-loop infusion system (MCCLIS) has been developed that provides stable intraoperative levels of partial neuromuscular blockade. Complete neuromuscular blockade interferes with intraoperative motor-evoked potential (MEP) monitoring used for patients undergoing surgical procedures that place them at risk for spinal cord ischemia. Nine patients were studied during which the MCCLIS maintained stable levels of partial neuromuscular blockade and allowed transcranial magnetic motor-evoked potential (TcM-MEP) monitoring during thoracoabdominal aortic aneurysmectomy. The use of TcM-MEP for monitoring intraoperative spinal cord function was balanced against surgical considerations for muscle relaxation with 80% to 90% neuromuscular blockade fulfilling each requirement. Intraoperative adjustment of partial neuromuscular blockade to facilitate TcM-MEP monitoring was also possible with the MCCLIS. The MCCLIS should allow for further investigation into the sensitivity, specificity, and predictability of TcM-MEP monitoring for any patient at risk for intraoperative spinal cord ischemia including those undergoing thoracoabdominal aortic aneurysmectomy.

The technique and efficacy of axillary catheter analgesia as an adjunct to distraction elbow arthroplasty: A prospective study.

A prospective study was done on 99 consecutive patients who underwent distraction elbow arthroplasty and postoperative continuous passive motion from 1986 to 1992. Postoperative axillary catheter infusion in 73 patients was compared with patient-controlled analgesia-opioid treatment in 26 patients. Group comparisons for analgesia, complications, time to initiation, and tolerance of postoperative continuous passive motion were made. The postoperative axillary catheter infusion group was more comfortable and had better postoperative continuous passive motion compliance than did the patient-controlled analgesia-opioid group. The postoperative axillary catheter infusion group had a 2.7% (2 of 73) incidence of respiratory toxicity. There was an 11.5% (3 of 26) incidence of respiratory depression in the patient-controlled analgesia-opioid group. Final arc range of motion was similar between postoperative axillary catheter infusion and patient-controlled analgesia-opioid groups. The data support the hypothesis that postoperative axillary catheter infusion provides better patient comfort and earlier postoperative continuous passive motion initiation, but they do not show additional improvement in the final arc of motion after distraction elbow arthroplasty as compared with a patient-controlled analgesia-opioid treatment group.

Empathic accuracy in the interactions of male friends versus male strangers.

In unstructured interactions, male friends were found to be more accurate than male strangers in inferring each other's thoughts and feelings. Plausible reasons for this difference were that friends (a) interacted more and exchanged more information, (b) had more similar personalities and therefore more rapport with each other, and (c) had more detailed knowledge of each other's lives. Data confirmed that the friends did indeed interact more and were more similar in their sociability than the strangers; however, these differences did not account for the friends' greater empathic accuracy. Instead, this was primarily attributable to a difference in knowledge structures, namely, the friends' ability to accurately read their partners' thoughts and feelings about imagined events in another place or time.

Molecular and developmental characterization of the heat shock cognate 4 gene of Drosophila melanogaster.

The Drosophila heat shock cognate gene 4 (hsc4), a member of the hsp70 gene family, encodes an abundant protein, hsc70, that is more similar to the constitutively expressed human protein than the Drosophila heat-inducible hsp70. Developmental expression revealed that hsc4 transcripts are enriched in cells active in endocytosis and those undergoing rapid growth and changes in shape.

In situ/invasive cervical cancer ratios: impact of cancer education and screening.

A study of cancer incidence rates in Newark, NJ showed an increase in invasive cervical cancer among black women between 1970 and 1976. The incidence was substantially higher than that found in the Third National Cancer Survey and the Surveillance Epidemiology End Results Program. The ratio of in situ/invasive cervical cancer incidence among black women was 1:1, far below the national ratio of about 3:1. It seemed that the high-risk population in Newark might not be having regular Papanicolaou smears. From 1976 through 1981, intensive educational and cervical cancer screening programs were carried out in the city of Newark. A review was made of cervical cancer cases (1976-1981) reported to the tumor registries or recorded on discharge diagnoses in hospitals where Newark residents are known to have sought care. The findings showed that the ratio of in situ to invasive cervical cancer had increased dramatically: the overwhelming majority of cancers were found to be in situ carcinomas. The improvement was far more impressive among black women than among white women. Since the intervention campaign had been directed primarily toward the black female population, a group at extremely high risk, this suggested that efficacy of the intervention program.

Expression and localization of Drosophila melanogaster hsp70 cognate proteins.

Monoclonal antibodies have been used to identify three proteins in Drosophila melanogaster that share antigenic determinants with the major heat shock proteins hsp70 and hsp68. While two of the proteins are major proteins at all developmental stages, one heat shock cognate protein, hsc70, is especially enriched in embryos. hsc70 is shown to be the product of a previously identified gene, Hsc4. We have examined the levels of hsp70-related proteins in adult flies and larvae during heat shock and recovery. At maximal induction in vivo, hsp70 and hsp68 never reach the basal levels of the major heat shock cognate proteins. Monoclonal antibodies to hsc70 have been used to localize it to a meshwork of cytoplasmic fibers that are heavily concentrated around the nucleus.

Reliability of criteria for predicting persistent or recurrent sepsis.

We reviewed the charts of 2,567 patients from 11 prospective clinical trials of antibiotic therapy for surgical infection to identify reliable predictors of sepsis eradication. Particular attention was paid to temperature, blood cell counts, renal and hepatic function tests, arterial gases, and clotting factors, both at the termination of parenteral antibiotic administration as well as at patient discharge from the hospital. On the discontinuation of antibiotic therapy, sepsis recurred in 19% of the patients who had a normal rectal temperature, in 3% of the patients if the rectal temperature and WBC count were normal, but in no patient when both the temperature and WBC count were normal and the differential blood smear contained less than 73% granulocytes and less than 3% immature forms. Rates for recurrent sepsis, once antibiotic therapy was discontinued for more than 48 hours, were 8%, 2%, and 0%, respectively, for the same criteria at hospital discharge.

Driving performance of retinitis pigmentosa patients.

The driving performance of 42 patients with retinitis pigmentosa (RP) was compared with that of 87 control subjects. Although the patients with RP were involved in more isolated road accidents than the control group, 50% were not involved in any accident over the most recent 5-year driving period. When the associations between driving performance and case/control status were examined (the number of driving hours per week and driving years being taken into account), differences in the number of accidents between the 2 groups were significant because of a disproportionately high number of accidents caused by a subgroup of female RP patients. No significant correlations were apparent between central visual efficiency or peripheral field efficiency and number of road accidents.

The platelet estimate: is it being ignored?

A study was undertaken to determine whether the platelet estimate included in the routine CBC is being utilized as a guide to ordering quantitative platelet counts. Requisition slips and results were reviewed for a one-month period. The data showed that platelet estimates were not being used properly. In fact, much of the time the platelet count was being ordered with the CBC. The smear estimate and quantitative count agreed in 96% of specimens. The platelet count is a costly procedure and should be reserved for those occasions when the platelet estimate is abnormal.