Quantitative comparison of intrabrain diffusion in adults and preterm and term neonates and infants

OBJECTIVE: Quantitative measurements of mean water diffusivity (D(av)) were made in human neonates, infants, and adults to assess changes in brain tissue that occur with maturation. SUBJECTS AND METHODS: Values of D(av) were obtained by calculating the average of the diffusion measurements made with diffusion-sensitizing gradients placed along three orthogonal directions. The mean diffusivity, a rotationally invariant determination of apparent diffusion coefficient, was measured in five healthy prematurely born neonates and infants, in 10 healthy term neonates and infants, and in five adults. RESULTS: Values of D(av) were found to decrease with maturation in most parts of the brain. In prematurely born neonates and infants with a postmenstrual age (postgestastional age + postnatal age) under 36 weeks, the average value of D(av) in frontal white matter was 1.90 x 10(-3) mm2 sec(-1). The corresponding value was measured as 1.62 x 10(-3) mm2 sec(-1) in neonates and infants born at term with a postnatal age of no more than 43 days and 0.79 x 10(-3) mm2 sec(-1) in the adult brain. CONCLUSION: Values of D(av) are known to decrease in neonates and young infants in the period immediately after ischemic insult. This decrease and the associated increase in signal intensity seen on diffusion-weighted imaging have been used to monitor ischemic brain injury in neonates and infants. Therefore, the decrease in D(av) that occurs with maturation, which we report in this study, must be considered if quantitative diffusion measurements are used to assess ischemic neonatal brain injury.     

Chronic oral treatment with 13-cis-retinoic acid (isotretinoin) or all-trans-retinoic acid does not alter depression-like behaviors in rats.

Oral treatment with the anti-acne drug Accutane (isotretinoin, 13-cis-retinoic acid) has been associated with suicide ideation and depression. Here, depression-like behaviors (i.e., behavioral despair and anhedonia) were quantified in adult Sprague-Dawley rats gavaged daily beginning at postnatal day (PND) 82 with 13-cis-RA (7.5 or 22.5 mg/kg) or all-trans-retinoic acid (10 or 15 mg/kg ). Tested at PND 130-131 in the Forced Swim Test, 7.5 mg/kg 13-cis-RA marginally decreased immobility and slightly increased climb/struggle durations whereas neither all-trans-retinoic acid group differed from controls. Voluntary saccharin solution (0.03%) intake at PND 102-104 and PND 151-153 was not different from controls in any treated group, although all RA-treated groups had lower intakes. Swim speed in a water maze at PND 180 was similar across groups, indicating no RA-induced differences in physical ability. Open field activity was mildly decreased at PND 91 in 7.5 mg/kg-treated males only, but it was within the control range at PND 119, 147, and 175. Thus, at serum levels similar to those in humans receiving the drug, chronic 13-cis-RA treatment did not severely affect depression-like behaviors in rats. These data do not substantiate the hypothesis of 13-cis-RA-induced depression.     

Abstinence-induced changes in self-report craving correlate with event-related FMRI responses to smoking cues.

Drug cues have been shown to activate brain regions involved in attention, motivation, and reward in addicted users. However, as studies have typically measured responses in only one state (ie drug abstinence), it is unclear whether observed activations represent amplification by abstinence or stable responses. Thus, the present study was designed to evaluate the stability of event-related responses to visual drug cues in dependent smokers (n=13) using event-related functional magnetic resonance imaging measures. Imaging was conducted following smoking as usual and following overnight abstinence, and self-reported craving measures were obtained before, during, and after scanning. Analysis of hemodynamic response (HDR) amplitudes in each of 13 regions of interest revealed larger responses to smoking compared to control cues in ventral anterior cingulate gyrus (vACG) and superior frontal gyrus. Responses to smoking cues in these and all other regions revealed no effects of abstinence/satiety, thus supporting the notion that cue-elicited brain responses are relatively stable. However, while the abstinence manipulation did not alter group-level responses to smoking cues, at the individual level, abstinence-induced changes in craving (abstinence minus satiety) were positively correlated with changes in HDR amplitude to smoking cues in frontal regions including left inferior frontal gyrus, left vACG, and bilateral middle frontal gyrus. These results suggest that brain responses to smoking cues, while relatively stable at the group level following short-term abstinence, may be modulated by individual differences in craving in response to abstinence-particularly in regions subserving attention and motivation.     

Significantly higher pathologic complete remission rate after neoadjuvant therapy with trastuzumab, paclitaxel, and epirubicin chemotherapy: results of a randomized trial in human epidermal growth factor receptor 2-positive operable breast cancer.

PURPOSE: The objective of this study was to determine whether the addition of trastuzumab to chemotherapy in the neoadjuvant setting could increase pathologic complete response (pCR) rate in patients with human epidermal growth factor receptor 2 (HER2) -positive disease. PATIENTS AND METHODS: Forty-two patients with HER2-positive disease with operable breast cancer were randomly assigned to either four cycles of paclitaxel followed by four cycles of fluorouracil, epirubicin, and cyclophosphamide or to the same chemotherapy with simultaneous weekly trastuzumab for 24 weeks. The primary objective was to demonstrate a 20% improvement in pCR (assumed 21% to 41%) with the addition of trastuzumab to chemotherapy. The planned sample size was 164 patients. RESULTS: Prognostic factors were similar in the two groups. After 34 patients had completed therapy, the trial's Data Monitoring Committee stopped the trial because of superiority of trastuzumab plus chemotherapy. pCR rates were 25% and 66.7% for chemotherapy (n = 16) and trastuzumab plus chemotherapy (n = 18), respectively (P = .02). The decision was based on the calculation that, if study continued to 164 patients, there was a 95% probability that trastuzumab plus chemotherapy would be superior. Of the 42 randomized patients, 26% in the chemotherapy arm achieved pCR compared with 65.2% in the trastuzumab plus chemotherapy arm (P = .016). The safety of this approach is not established, although no clinical congestive heart failure was observed. A more than 10% decrease in the cardiac ejection fraction was observed in five and seven patients in the chemotherapy and trastuzumab plus chemotherapy arms, respectively. CONCLUSION: Despite the small sample size, these data indicate that adding trastuzumab to chemotherapy, as used in this trial, significantly increased pCR without clinical congestive heart failure.     

Randomized phase II study of two doses of gefitinib in hormone-refractory prostate cancer: a trial of the National Cancer Institute of Canada-Clinical Trials Group.

PURPOSE: Overexpression of the epidermal growth factor receptor has been demonstrated in advanced prostate cancer and is associated with a poor outcome. A multi-institutional, randomized, phase II study was undertaken by the National Cancer Institute of Canada-Clinical Trials Group to evaluate the efficacy and toxicity of two doses of oral gefitinib in patients with minimally symptomatic, hormone-refractory prostate cancer (HRPC). PATIENTS AND METHODS: Between July and November 2001, 40 patients with HRPC and increasing prostate-specific antigen (PSA) or progression in measurable disease who had not received prior chemotherapy were randomly assigned to 250 mg (n = 19) or 500 mg (n = 21) oral gefitinib daily continuously. The primary end points were PSA response rate and objective measurable response. Functional Assessment of Cancer Therapy Prostate Cancer Subscale (FACT-P) quality-of-life questionnaires were completed at baseline and during treatment. RESULTS: None of the patients demonstrated a PSA or objective measurable response. Five (14.3%) of 35 assessable patients had stable PSA (one patient at 250 mg and four patients at 500 mg), and five patients (14.3%) had a best response of stable disease (duration, 2.5 to 16.8 months). No significant effect on the rate of increase in PSA was seen. The most common drug-related nonhematologic toxicities observed were grade 1 to 2 diarrhea (250 mg, 65%; 500 mg, 56%), fatigue (250 mg, 29%; 500 mg, 33%), and grade 1 to 2 skin rash (250 mg, 24%; 500 mg, 39%). FACT-P scores decreased during treatment, indicating worsening of symptoms compared with baseline. CONCLUSION: Gefitinib did not result in any responses in PSA or objective measurable disease at either dose level. Gefitinib has minimal single-agent activity in HRPC.     

Experience of combined endoscopic percutaneous stenting with ultrasound guidance for drainage of pancreatic pseudocycts

The therapeutic options for treatment of pancreatic pseudocysts are numerous. We report our experience of combined endoscopic and ultrasound guided percutaneous stenting for pancreatic pseudocysts. Data were prospectively collected for 20 consecutive patients. All patients had undergone a standard technique of combined endoscopic and ultrasound guided percutaneous placement of double J stents, between a pancreatic pseudocyst and the stomach. Patients age ranged between 25 and 84 years. Thirteen of the pseudocysts were due to acute pancreatitis and 7 were due to chronic pancreatitis. The duration of the combined procedure was mean 50 min (range 30-95 min). The length of hospital stay was mean 5 days (range 2-77 days. Only two patients suffered postoperative complications; one was re-admitted 2 weeks following stenting with acute cholecystitis, the other suffering a perforated duodenal ulcer 3 weeks after stenting. There were two failures early in the series, both due to stent migration, these stents were of a small size, (4.7 French). Following this the stent size was increased to at least 7 French, no further failures occurred. There was no operative mortality for the series. Follow-up ranged between 6 months and 5 years. We conclude that a combined percutaneous and endoscopic cyst-gastrostomy stent is a safe and effective treatment for patients with suitably placed pseudocysts.     

Bilobed oblong porous coated acetabular components in revision total hip arthroplasty

Thirty-eight oblong bilobed noncustom uncemented, porous-coated titanium acetabular components were used to reconstruct failed hip arthroplasties with large superior segmental acetabular bone deficiencies. No structural bone grafts were used. All patients were followed up for 2 to 5 years (mean, 3 years) after the operation. One patient (whose socket rested primarily on a structural bone graft from a previous procedure) had revision surgery for acetabular loosening. No other patients have had revision surgery or had another ipsilateral hip operation. At latest followup, 35 patients had no or mild pain and two patients had moderate pain. Two implants migrated more than 2 mm in the first year, then stabilized. On the latest radiographs, two implants had bead shedding, but there was no measurable migration or change in position. For selected patients with large superolateral acetabular bone deficiencies, this implant facilitated a complex reconstruction, provided good clinical results, and showed satisfactory stability at early to midterm followup in most patients.     

A novel function of bamboo extract in relieving lipotoxicity

Lipotoxicity is closely related to the etiology and complications of type 2 diabetes mellitus. This study investigated the protective effect of an extract from bamboo Phyllostachys edulis against palmitic acid (PA)-induced lipoapoptosis. The lipo-detoxification function of the bamboo extract (BEX) was evaluated using cell culture models. Cell viability was measured by MTT assay and cell apoptosis was monitored by Annexin V staining. Cellular uptake of fluorescent free fatty acid (FFA) analog was measured by flow cytometry. Protein levels of total protein kinase B (Akt) and phosphorylated Akt (p-Akt) were measured by western blotting. The results show that co-incubating BEX with mouse myoblast C2C12 cells had no effect on the cellular uptake of FFA, but dramatically decreased PA-induced cell apoptosis and protected cell viability. A similar antilipotoxicity effect of BEX was observed in other mammalian cells. BEX significantly decreased the protein levels of both Akt and p-Akt in C2C12 cells under normal cell culture conditions but not under lipotoxic conditions, indicating the regulatory effect of BEX on cell signaling pathways and its response to a high FFA environment. This study demonstrated a novel function of bamboo extract in preventing lipotoxicity in mammalian cells, implicating a promising phytotherapeutic approach for lipo-detoxification.     

SRS-FISH: A high-throughput platform linking microbiome metabolism to identity at the single-cell level

One of the biggest challenges in microbiome research in environmental and medical samples is to better understand functional properties of microbial community members at a single-cell level. Single-cell isotope probing has become a key tool for this purpose, but the current detection methods for determination of isotope incorporation into single cells do not allow high-throughput analyses. Here, we report on the development of an imaging-based approach termed stimulated Raman scattering–two-photon fluorescence in situ hybridization (SRS-FISH) for high-throughput metabolism and identity analyses of microbial communities with single-cell resolution. SRS-FISH offers an imaging speed of 10 to 100 ms per cell, which is two to three orders of magnitude faster than achievable by state-of-the-art methods. Using this technique, we delineated metabolic responses of 30,000 individual cells to various mucosal sugars in the human gut microbiome via incorporation of deuterium from heavy water as an activity marker. Application of SRS-FISH to investigate the utilization of host-derived nutrients by two major human gut microbiome taxa revealed that response to mucosal sugars tends to be dominated by Bacteroidales, with an unexpected finding that Clostridia can outperform Bacteroidales at foraging fucose. With high sensitivity and speed, SRS-FISH will enable researchers to probe the fine-scale temporal, spatial, and individual activity patterns of microbial cells in complex communities with unprecedented detail.

With the rapid advances in both genotyping and phenotyping of single cells, bridging genotype and phenotype at the single-cell level is becoming a new frontier of science (1). Methods have been developed to shed light on the genotype–metabolism relationship of individual cells in a complex environment (2, 3), which is especially relevant for an in-depth understanding of complex microbial communities in the environment and host-associated microbiomes. For functional analyses of microbial communities, single-cell isotope probing is often performed in combination with nanoscale secondary ion mass spectrometry (NanoSIMS) (4–7), microautoradiography (MAR) (8, 9), or spontaneous Raman microspectroscopy (10–12) to visualize and quantify the incorporation of isotopes from labeled substrates. These methods can be combined with fluorescence in situ hybridization (FISH) using ribosomal ribonucleic acid (rRNA)-targeted probes (13), enabling a direct link between metabolism and identity of the organisms. In addition, Raman-activated cell sorting has been recently developed using either optical tweezers or cell ejection for downstream sequencing of the sorted cells (14–16). While these approaches have expanded the possibilities for functional analyses of microbiome members (17), all of the aforementioned methods suffer from extremely limited throughput. Consequently, only relatively few samples and cells per sample are typically analyzed in single-cell stable isotope probing studies, hampering a comprehensive understanding of the function of microbes in their natural environment.To overcome the limited throughput of Raman spectroscopy, coherent Raman scattering microscopy based on coherent anti-Stokes Raman scattering (CARS) or stimulated Raman scattering (SRS) has been developed (18, 19). Compared with CARS, the SRS signal is free of the electronic resonance response (20) and is linear to molecular concentration, thus permitting quantitative mapping of biomolecules (21, 22). Both CARS and SRS microscopy have successfully been applied for studying single-cell metabolism in eukaryotes (23–26). In a label-free manner, SRS imaging has led to the discovery of an aberrant cholesteryl ester storage in aggressive cancers (27, 28), lipid-rich protrusions in cancer cells under starvation (29), and fatty acid unsaturation in ovarian cancer stem cells (30) and more recently, in melanoma (31, 32). CARS and SRS have also been harnessed to explore lipid metabolism in live Caenorhabditis elegans (33–36). Combined with stable isotope probing, SRS microscopy has allowed the tracing of glucose metabolism in eukaryotic cells (37, 38) and the visualization of metabolic dynamics in living animals (25). Recently, SRS was successfully applied to infer antibiotic resistance patterns of bacterial pure cultures and heavy water (D2O) metabolism (39). Yet, SRS microscopy has not been adapted for studying functional properties of members of microbiomes as SRS itself lacks the capability of identifying cells in a complex community.Here, we present an integrative platform that exploits the advantages of SRS for single-cell stable isotope probing together with two-photon FISH for the identification of cells in a high-throughput manner. To deal with the challenges in detecting low concentrations of metabolites inside small cells with diameters around 1 µm, we have developed a protocol that maximizes the isotope label content in cells and exploits the intense SRS signal from the Raman band used for isotope detection.Conventionally, FISH is performed separately by one-photon excited fluorescence microscopy (40). To enhance efficiency, we developed a system that implements highly sensitive SRS metabolic imaging with two-photon FISH using the same laser source. These efforts collectively led to a high-throughput platform that enables correlative imaging of cell identity and metabolism at a speed of 10 to 100 ms per cell. In comparison, it takes about 20 s to record a Raman spectrum from a single cell in a conventional spontaneous Raman FISH experiment (41, 42).Our technology enabled high-throughput analysis of single-cell metabolism in the human gut microbiome. In the human body, microbes have been shown to modulate the host’s health (43, 44). Analytical techniques looking into their activities and specific physiologies (i.e., phenotype) as a result of both genotype and the environment provide key information on how microbes function, interact with, and shape their host. As a proof of principle, we used stimulated Raman scattering–two-photon fluorescence in situ hybridization (SRS-FISH) to track the incorporation of deuterium (D) from D2O into a mixture of two distinct gut microbiota taxa. Incorporation of D from D2O into newly synthesized cellular components of active cells, such as lipids and proteins, occurs analogously to incorporation of hydrogen from water during the reductive steps of biosynthesis of various cellular molecules (10, 45, 46). Importantly, D incorporation from D2O has been shown to be reliable to track metabolic activity of individual cells within complex microbial communities in response to the addition of external substrates (10, 17, 47). When microbial communities are incubated in the presence of D2O under nutrient-limiting conditions, individual cells display only minimal activity and only minor D incorporation (11, 17, 47). In contrary, when cells are stimulated by the addition of an external nutrient, cells that can metabolize this compound become active and incorporate D into macromolecules, which lead to the presence of C-D bonds into the cell’s biomass. Consequently, D incorporation from D2O can be combined with techniques able to detect C-D signals, such as Raman-based approaches, and to track metabolic activity at the single-cell level in response to a variety of compounds. Here, we show that SRS-FISH enables fast and sensitive determination of the D content of individual cells while simultaneously unveiling their phylogenetic identity. We applied this technique to complex microbial communities by tracking in situ the metabolic responses of two major phylogenetic groups of microbes in the human gut (Bacteroidales and Clostridia spp.) and of a particular species within each group to supplemented host-derived nutrients. Our study revealed that 1) Clostridia spp. can actually outperform Bacteroidales spp. at foraging on the mucosal sugar fucose and shows 2) a significant interindividual variability of responses of these major microbiome taxa toward mucosal sugars. Together, our results demonstrate the capability of SRS-FISH to unveil the metabolism of particular microbes in complex communities at a throughput that is two to three orders of magnitude higher than other metabolism identity bridging tools, therefore providing a valuable multimodal platform to the field of single-cell analysis.     

Gregor Johann Mendel and Modern Evolutionary Biology: Mendel and Darwin

Evolution by natural selection is an explicitly genetic theory. Darwin recognized that a working theory of inheritance was central to his theory and spent much of his scientific life seeking one. The seeds of his attempt to fill this gap, his “provisional hypothesis” of pangenesis, appear in his notebooks when he was first formulating his evolutionary ideas. Darwin, in short, desperately needed Mendel. In this paper, we set Mendel’s work in the context of experimental biology and animal/plant breeding of the period and review both the well-known story of possible contact between Mendel and Darwin and the actual contact between their ideas after their deaths. Mendel’s contributions to evolutionary biology were fortuitous. Regardless, it is Mendel’s work that completed Darwin’s theory. The modern theory based on the marriage between Mendel’s and Darwin’s ideas as forged most comprehensively by R. A. Fisher is both Darwin’s achievement and Mendel’s.