Further, the T2 relaxation of the semisolid macromolecular pool , a parameter specific to MT, was found to be significantly different (p < 0.01). Also significantly different were the rNOE contributions associated with methine groups at −0.9 ppm with a saturation B1 of 0.5 µT (p < 0.01) and with other aliphatic groups at −3.3 ppm with 0.5 and 2 µT (both p < 0.05). Independently, using a live-cell metabolic assay, normal cells were found to have a greater metabolic rate than radioresistant ones. Thus, MRI provides a novel, in vivo method to quantify the metabolic rate of tumours and predict their radiosensitivity.The ability of MRI to differentiate between normal and radioresistant cancer was investigated in prostate tumour xenografts in mice. Specifically, the process of magnetization exchange between water and other molecules was studied. It was found that magnetization transfer from semisolid macromolecules (MT) and chemical exchange saturation transfer (CEST) combined were significantly different between groups ( Recurrent tumours, which have survived radiation , tend to display an aggressive phenotype including increased proliferation, clinically manifest as larger tumours that are typically associated with lymph node metastases, and generally have a worse prognosis7. Indeed, up to a third of patients with recurrent prostate cancer will die from their cancer9.Prostate cancer is the most prevalent non-skin cancer in men and one of the leading causes of cancer death. Both surgery and radiotherapy are well established modalities used in its treatment. Unfortunately, almost one-third of high-risk prostate cancer patients develop recurrence following external beam radiation treatment as assessed by rising prostate-specific antigen levelsWe believe that development of improved detection of prostate cancer radioresistance is essential for further improving patient outcomes. The non-invasive detection of radioresistant prostate cancer through quantitative MRI will allow rapid and tailored treatment decisions to be made, such as the addition of radiosensitizers or, alternatively, the use of surgical resection in place of radiation treatment.11 (DW-MRI), dynamic contrast-enhanced MRI12 (DCE-MRI), and positron emission tomography14 (PET). Some of these techniques were able to detect radioresistance as early as a few weeks or a month. Confounding factors include sensitivity to multiple tissue features such as microstructural geometry and permeability which may not be specific to radiation resistance15. Furthermore, DCE-MRI requires the injection of contrast agent16, and PET exposes the patient to ionizing radiation and has a limited resolution17.Previous studies have assessed tumour response to therapy using diffusion-weighted MRI18 (CEST) and the relayed nuclear Overhauser effect19 (rNOE) are promising magnetic resonance contrast mechanisms that are sensitive to metabolism20, can provide contrast without an exogenous contrast agent, and can potentially predict tumour response before treatment21. They are measured using magnetization transfer-prepared pulse sequences, which are sensitive to the exchange of magnetization between the hydrogen nuclei in water and other molecules. CEST is the physical exchange of hydrogen atoms in chemical groups in dissolved proteins  with water. rNOE is the exchange of magnetization intramolecularly through space between chemical groups  with other hydrogen nuclei, which then undergo CEST27. In addition to CEST and rNOE, magnetization transfer-prepared pulse sequences can measure the exchange of magnetization between semisolid macromolecules (mostly lipid bilayers) and water, which is termed magnetization transfer29 (MT).Chemical exchange saturation transferB1) across the various frequencies of the exchanging molecules. The ratio of this reduced water signal (S) to the signal without saturation (S0) is calculated for each frequency offset, and from this the magnetization transfer ratio (MTR) can be calculated as: 1 – S/S0. In addition to the exchange rates of magnetization between hydrogen nuclei in the semisolid macromolecular, CEST, and rNOE pools with those in water, the MTR is also sensitive to the sizes and longitudinal and transverse relaxation times  of each pool. It is also common to present the CEST data in a form of a “Z-spectrum”, which is a plot of measured water signal as a function of saturation pulse frequency offset (Δω) acquired over multiple excitations.In magnetization transfer-prepared pulse sequences, magnetization is reduced by a radiofrequency saturation pulse  ones and, consistent with this, discovered that they exhibit different MT, CEST, and rNOE effects in vivo – MRI contrasts that are sensitive to the chemical environment. In this work, we show that normal and radioresistant tumours in an animal model can be differentiated by MTR and isolate the underlying MT and rNOE contributions.A radioresistant prostate cancer cell line has been developed to further investigate radioresistancein vivo. We have also analyzed the contributions of several MRI effects to the CEST spectra which allowed us to determine which of the many MRI processes  are the major contributor for the observed changes between parental and radioresistant tumour xenografts.In this work, the CEST effect was measured for 7 parental and 6 radioresistant DU145 prostate tumours xenografts T2-weighted image  were seen on T1 and T2 maps. Their Z-spectra were also substantially different than those of the other parental and all the radioresistant tumours  and 2 µT (orange). The mean Z-spectra for parental and radioresistant tumours, although similar in shape, exhibited significant differences, which are more visible in the enlarged plots in Fig. B1. The MTR with a saturation B1 of 0.5 µT at Δω = −0.9 ppm  for parental and radioresistant tumours were 2260 ± 100 and 2300 ± 70 ms, respectively, while observed T2s (from the inversion recovery images and WASSR spectrum) were 61 ± 7 and 64 ± 4 ms, respectively, neither of which was significantly different between groups. T1 and MTR histograms are in Supplementary Fig. The averaged Z-spectra of the remaining homogeneous parental and radioresistant tumours are shown in Fig. B1s of 3 and 6 µT, were fitted to a two-pool MT model  between the parental and radioresistant groups. These fitted parameters were used to extrapolate the MT model Z-spectra to the CEST- and rNOE-sensitive saturation B1s of 0.5 and 2 µT. A schematic is in Supplementary Fig. B1 of 0.5 µT , in combination with the modelled MT contribution extrapolated to these B1s, separated out the relative contributions of CEST and rNOE in both tumour types  at a single, low saturation B1 Fig. , which i29, which is a function of T1 and T2. However, the relaxation values of parental and radioresistant tumours were similar indicating that the differences in the MTR originated primarily from semisolid macromolecular MT and rNOE.Both the semisolid macromolecular and aliphatic rNOE pools contributed to the difference in MTR between the two tumour groups Table . The abi31 (MTRasym), a common metric to identify CEST contrast. It is calculated by subtracting the Z-spectrum at positive offsets from the corresponding negative offsets. MTRasym combines the contrast from CEST and rNOE, which in this case actually decreased the difference between parental and radioresistant groups.This dual contribution to the MTR signal was also the reason we do not use MTR asymmetryin vitro observation that radioresistant tumour cells had lower metabolism than the parental ones  than parental (8.44 ± 0.29 µs). T2,B has been found to vary with tissue type33, and changes in T2,B have also been attributed to the degree of cross-linking of molecules . Decreased T2,B has been observed in spinal cord neuropathy where it was interpreted as a change in macromolecular structure of myelin35. We believe that this is the first observation of this phenomenon in cancer tissue where it may indicate slight changes in the rigidity of cell membrane lipids which are believed to be responsible for MT effects in tumours.Of the semisolid macromolecular MT parameters, the only one showing significant difference was the semisolid macromolecular relaxation time et al.36 in several respects. First, we acquired Z-spectra at logarithmically spaced frequency offsets from 300 to 3 ppm, whereas Heo et al. acquired Z-spectra with linearly spaced offsets from 21 to −21 ppm, excluding the range from 7 to −7 ppm to avoid CEST and rNOE effects. Note that the semisolid macromolecular MT Z-spectrum has a feature around 50 ppm37 that necessitates data collection beyond 21 ppm. Second, we simultaneously fitted the Z-spectra and T1 map to the two-pool MT model, whereas Heo et al. fitted the Z-spectra alone to yield parameters lumped together with the T1 of the water pool and then isolated the parameters using the T1 map. We feel that simultaneous analysis of all the data allowed the fitting algorithm to better constrain the parameter estimates. Third, after subtracting the semisolid macromolecular MT contribution from the low B1 Z-spectra, we compared the CEST- and rNOE-only MTRs, whereas Heo et al. fitted the Bloch–McConnell38 magnetization exchange equations to estimate the pool sizes, exchange rates, and T2s of the amide (3.5 ppm) and rNOE pools (−2.5 to −5 ppm). Each has a unique advantage. The benefit of fitting the Bloch–McConnell equations is that the parameters are B1-independent. The benefit of comparing CEST- and rNOE-only MTRs is that it does not require the collection of full Z-spectra at lower B1. We attempted fitting of the Bloch–McConnell equations with amide (3.5 ppm), guanidinium (2 ppm), and rNOE pools (−3.3 ppm) to the CEST- and rNOE-only Z-spectra as well as all the data , but there was structure in the residuals and this was left to future work. Some misfitting is also seen in the work by Heo et al. , possibly due to the lack of a pool in their model at 2 ppm.Our isolation of the CEST and rNOE contributions differed from the extrapolated semi-solid magnetization transfer reference (EMR) method by Heo 39 it appears as one broad peak, instead of multiple peaks. Phantom studies would help, but it is difficult to produce a simple phantom with an aliphatic rNOE pool. Ex vivo rNOE has been measured using protein-free brain lipids extracted from mice, but not in synthetic liposomes40. It has also been studied using water-exchange (WEX) filter spectroscopy experiments, which showed that the rNOE process exchanges magnetization with water over hundreds of milliseconds, much slower than the CEST exchange rate (<100 ms)19.Another challenge to the modelling and interpretation of CEST and rNOE data is that our understanding of the rNOE contributions is limited. rNOE is thought to be composed of several peaks corresponding to different aliphatic groups. However, even at the extremely high field of 21.1 TT2-weighted structural image gave better contrast between muscle, tumour, and liquid voxels, which was needed for accurate tumour segmentation. However, a T2 map would have the desirable property over a T2-weighted image of not being TR- and TE-dependent. Unfortunately, a T2 map was not part of the original imaging protocol and was generated from the T1 and WASSR data instead, which is not conventionally done. In our opinion, a more standard CPMG-calculated42T2 map would be free of any potential cumulative errors from the T1 and WASSR scans and does not take long to acquire for a single slice. A CPMG sequence will be included in future studies.The 43 demonstrated that pH is a function of the expression:Mz is the measured CEST contrast, M0 is the contrast in the absence of radiofrequency saturation or under control saturation, and the sites refer to different chemical groups that are saturated. McVicar et al.44 showed that, when Site 2 is assigned to amine (2.75 ppm) and Site 1 to amide (3.5 ppm), Eq. 45 reported a similar finding with guanidinium (2 ppm) in place of amine. Eq. B1s of 0.5 and 2 µT with Site 2 assigned to guanidinium and Site 1 to amide and M0 defined as unity. Eq. There is likely a difference in pH between the two tumours types to which CEST may be sensitive. Although we have not assayed pH or reactive oxygen species levels in our tumours to date, based on the reduced oxygen consumption rate of the radioresistant cells relative to parental cells, this could translate to reduced hypoxia and less acidosis in the radioresistant cells. It is also possible that, given tumour heterogeneity, the exported lactate (resulting from a reduction in oxygen consumption and conversion of pyruvate to lactate by lactate dehydrogenase) is used as fuel by neighbouring cells, so an increase in acidosis may not be seen. This is further corroborated by the following CEST-derived metric. Ward and Balabanin vivo, the acquisition of all the Z-spectra, as performed in this work, would be too time consuming. Fortunately, to arrive at the conclusions presented requires much less data collection: two Z-spectra with high saturation B1 (3 and 6 µT) in order to extrapolate the semisolid macromolecular contribution to low saturation B1 (≈ 0.5 µT), a partial Z-spectrum with the lower saturation B1 around −0.9 ppm frequency offset to isolate the methine rNOE contribution and/or −3.3 ppm to isolate other aliphatic rNOE contributions or both (as single measurements at each of these offsets would be insufficient in the presence of B0 inhomogeneity), a WASSR scan for B0 correction, and a T1 map for two-pool MT model fitting.For applications T1). This simplifies quantitative MT modelling and future work in quantitative CEST because the effective saturation duration is 16 s (32 phase-encoding lines to reach the centre of k-space × 500 ms TR). Note that this should not to be confused with equilibrium magnetization reached after repeated TRs. In clinical imaging, faster readouts are used, where magnetization is not in steady state with respect to saturation46 and measurements at fewer frequency offsets are made, but these may be necessary trade-offs. Hardware limitations include lower RF amplitude and RF duty cycle, which may limit the saturation duration, require pulsed saturation (complicating modelling), and add dead time in each TR. All of this reduces image contrast relative to that from experiments on animal scanners. The clinical research magnetization transfer-prepared protocol for head imaging in our lab consists of single slice MT-prepared turbo field echo sequence with a saturation B1 of 0.5 µT made of four 242 ms block pulses, at 64 frequency offsets and 5 reference scans  and one average each with B1s of 3 and 5 µT at 11 frequency offsets ; WASABI47 for B0 and B1 mapping (1 min); a series of low flip angle fast field echo scans for T1 mapping (2 min); and a CPMG sequence for T2 mapping (1.5 min) for a total of 18 min. The trade-off between frequency offsets and number of slices for increased coverage should also be considered.We chose to use magnetization transfer-prepared FLASH to ensure that the net magnetization is in steady state with respect to saturation  generated by treatment of parental cells with radiation mimicking a clinical treatment schedule. Approximately 3 × 106 cells mixed in a 1:1 ratio with growth factor reduced Matrigel matrix  were injected in the right hind limbs of female athymic nude mice  and allowed to grow into tumours . Tumours were allowed to grow until they reached a volume of at least 100 mm3 measured using calipers every 1–4 days and calculated using the formula volume = length × width2/2 and until they were at least 34 days post-injection to allow time for cell differentiation. All experimental procedures in this study were approved by the Animal Care Committee of the Sunnybrook Research Institute, which adheres to the Policies and Guidelines of the Canadian Council on Animal Care and meets all the requirements of the Animals for Research Act of Ontario and the Health of Animals Act of Canada.Two cell lines were used in this study: a parental line, DU145 human prostate adenocarcinoma  and a radiation-resistant lineT2-weighted rapid acquisition with refocused echoes48  was used for prescribing the slice of interest, chosen to be at the thickest point of the tumour. B0-map-based shimming (map shim) was performed in an ellipsoidal volume enclosing the tumour in the slice of interest. Flip angle scale factor maps  was found to be within 6% of nominal. Thus, B1 correction was deemed unnecessary, given the time constraints. Z-spectra  composed of single slice images were calculated from magnetization transfer-prepared  fast low angle shot50  as in our previous work51. The cumulative saturation time when acquiring the centre of k-space is ≈ 16 s. Five Z-spectra were acquired: two spectra sensitive to the direct water saturation effect (DE), CEST, and MT contributions with radiofrequency saturation amplitudes, B1s, of 0.5 and 2 µT at 66 frequency offsets Δω  between ±5 ppm; two spectra mainly sensitive to the DE and magnetization transfer from semisolid macromolecules (MT) with saturation B1s of 3 and 6 µT at 11 logarithmically spaced offsets between 300 and 3 ppm; and one DE-sensitive water saturation shift referencing52 (WASSR) spectrum with a saturation B1 of 0.1 μT at 21 linearly spaced offsets between ±0.5 ppm. After every five Z-spectrum measurements, a reference scan at an offset of 667 ppm was interleaved for baseline correction. In addition, two initial and one final reference scans were acquired for each Z-spectrum. The scan time for the Z-spectra including reference scans with saturation B1s of 0.5 and 2 µT was 44 min/spectrum; 3 and 6 µT, 8.5 min/spectrum; and 0.1 µT, 15 min. Five inversion recovery RARE53 scans  were also acquired for a T1 map. The total acquisition time including scout and shimming per animal was 2.5 h.All tumours were scanned at 7 T  using a 86 mm inner diameter volume coil (T12053V3) for transmit and a 20 mm diameter loop surface coil (T115534) for receive. A 2D axial Anaesthesia in the animals was induced with 5% isoflurane in oxygen flowing at 1.5 L/min. 200 µL of saline solution was injected subcutaneously at the start of the scan to maintain hydration. Monitoring was performed with a temperature probe and respiratory pillow , both placed under the belly against the skin. Heating was supplied by a water bed system (same manufacturer). Skin temperature was kept at 36 °C and the isoflurane concentration adjusted around 1.75% such that the respiratory rate was maintained around 90 breaths/min.54 was applied to all Z-spectra. For each animal, images were registered using a rigid body transform to the first reference image with a saturation B1 of 0.5 µT. Z-spectrum images with less than 75% of the mean signal of the reference scan were considered to have insufficient signal-to-noise ratio (SNR) for the calculation of a transform matrix and were registered using the last matrix with sufficient SNR . To correct for baseline drift, the measurements of each Z-spectrum were normalized to a line fitted to the reference measurements interleaved with the Z-spectrum measurements (reference measurements are not shown). To correct for B0 inhomogeneity, which introduces a shift in the Z-spectrum along the frequency offset axis, the sum of two Lorentzians (corresponding to DE and semisolid macromolecular pools) was fitted to the Z-spectra with saturation B1s of 0.5 and 2 µT at offsets between ±0.5 ppm. The Z-spectra were re-centred to the peak position of the water-pool Lorentzian and linearly interpolated to the frequency offsets measured originally. Similarly, for the WASSR spectrum, a single Lorentzian was fitted for B0 correction (since there is negligible semisolid macromolecular MT for low B1 saturation amplitudes). This spectrum-wise B0 correction was chosen, instead of using WASSR to correct all spectra, in case B0 drifted during acquisition.The first reference scan of each Z-spectrum was discarded in case it was not in a steady state. The distribution of signal in a manually drawn background ROI was confirmed to be Rician and Rician noise bias correctionT1 map was calculated from the inversion recovery scans by fitting to the inversion recovery RARE signal equation53. Then, a T2 map was evaluated from the T1 map and WASSR spectrum , exchange rate from the semisolid macromolecular pool to the water pool (R), initial magnetization of semisolid macromolecular pool  relative to that of the water pool (defined as unity) and T2 of the semisolid macromolecular pool  and one calculated parameter: R1 of the water pool , evaluated from the T1 map and fitted parameters. These parameters, describing only the direct effect and magnetization transfer of semisolid macromolecules, were also compared between groups.The MTR contains contributions from the water, semisolid macromolecular (MT pool), CEST, and rNOE pools and are dependent on saturation 36, was employed. Two Z-spectra containing only semisolid macromolecular MT and water contributions were forward modelled using the estimated two-pool MT model parameters for saturation B1s of 0.5 and 2 µT .Finally, a method to isolate CEST and rNOE contributions, similar to the extrapolated semisolid magnetization transfer reference (EMR) techniquein vitro. Oxygen consumption rate profiles of the parental and radioresistant cells were measured (n = 8 wells per group) using a live cell metabolic assay platform  in the presence of 10 mM glucose, 1 mM pyruvate, and 2 mM glutamine56. The cells were subjected to serial injections of 2.0 µM oligomycin, which inhibited ATP synthase, but allowed the facilitated diffusion of protons or “proton leak” across the inner mitochondrial membrane; 1.0 µM p-trifluoromethoxy carbonyl cyanide phenylhydrazone (FCCP), which dissipated the inner mitochondrial membrane potential and maximized oxygen consumption; and 0.5 µM rotenone and antimycin A, which inhibited complexes I and III of the electron transport chain and stopped all mitochondrial respiration. This allowed the measurement of the oxygen consumption rate due to ATP production, the proton leak, and maximal respiration and the non-mitochondrial oxygen consumption rate, respectively, so that relative metabolic rate could be compared to relative CEST and rNOE contrast (an indirect measure of metabolism) between parental and radioresistant cells.An independent measurement of the metabolic rate of the two DU145 cell types was also performed The data that support the findings of this study are available from the corresponding author upon reasonable request.Supplementary Information


AbstractMegaselia Rondani is a large, poorly-known taxon whose species are found worldwide.The phorid fly genus Megaselia Rondani, M.simunorum, is described from both urban and rural sites in southern California. With a large area of white colour on the posterior part of the abdominal dorsum, it closely resembles the much more common species M.sulphurizona, but M.simunorum has distinctly thicker ventral setae on the abdomen and a differently-shaped white spot.A new species of  Megaselia Rondani has many difficult-to-separate species, but a few seem almost immediately identifiable, such as the common western North American species Megaseliasulphurizona Borgmeier. This species, although originally described from just eight specimens from California, Washington and Idaho, USA, is widespread within western USA and is one of the most abundant species collected in urban Los Angeles by the BioSCAN project (sulphurizona is loosely “yellow belt”). Borgmeier was working with air-dried specimens, in which colour can be distorted, however and we found that fresh specimens usually have a white spot. Regardless, until now, the identification of M.sulphurizona has been extremely straightforward.The enormous genus  project . The ori project  noted thM.sulphurizona and started to explore the variation within this species. We take this opportunity to describe our first, most distinctive, new white-spotted Megaselia that, based on its divergent abdominal structure, is apparently not closely related to M.sulphurizona (whose systematics we plan to study later).Amongst the many thousands of phorid flies captured by Malaise traps in the BioSCAN project were a few specimens of “another” white-spotted species. Furthermore, we found large differences in the extent of the white colour on the dorsum of The description of this species follows the reduced, table-based method we previously established . SpecimeBrown, Wong, and Hartopsp. n.urn:lsid:zoobank.org:act:D300CD48-1374-40A5-BC23-82EDF0E7467CType status:Holotype. Occurrence: catalogNumber: LACM ENT 366270; sex: male; Location: country: USA; stateProvince: California; county: Los Angeles; locality: Encino; verbatimCoordinates: 34.167°N, 118.513°W; verbatimCoordinateSystem: decimal degrees; decimalLatitude: 34.167; decimalLongitude: -118.513; Event: eventID: BioSCAN 18490; samplingProtocol: Malaise trap; Record Level: institutionCode: LACM; basisOfRecord: Preserved specimenType status:Paratype. Occurrence: recordedBy: B.Brown, G.Kung; sex: male; lifeStage: adult; Location: country: USA; stateProvince: California; county: Kern; locality: Wind Wolves Preserve; verbatimCoordinates: 34.956°N, 119.187°W; verbatimCoordinateSystem: decimal degrees; decimalLatitude: 34.956; decimalLongitude: -119.187; Event: samplingProtocol: Malaise trap; verbatimEventDate: 9-15.v.2018; Record Level: institutionID: LACM; institutionCode: LACM; basisOfRecord: PreservedSpecimenType status:Paratype. Occurrence: recordedBy: B.Brown, G.Hendler; sex: male; Location: country: USA; stateProvince: California; county: Los Angeles; verbatimLocality: Topanga Canyon; verbatimElevation: 250 m; minimumElevationInMeters: 250; maximumElevationInMeters: 250; decimalLatitude: 34.08; decimalLongitude: -118.60; Event: samplingProtocol: Malaise trap; verbatimEventDate: 7-14.iii.1994; Record Level: institutionID: LACM; basisOfRecord: PreservedSpecimenType status:Paratype. Occurrence: recordedBy: B.V.Brown; individualCount: 3; sex: male; lifeStage: adult; Location: country: USA; stateProvince: California; county: San Luis Obispo; locality: Los Osos,Pecho Willows; verbatimLocality: Pecho Willows; verbatimElevation: 5 m; decimalLatitude: 35.317; decimalLongitude: -120.853; Event: samplingProtocol: Malaise trap; verbatimEventDate: 2-8.vii.2017; eventRemarks: old field near bay; Record Level: institutionID: LACM; basisOfRecord: PreservedSpecimenType status:Paratype. Occurrence: recordedBy: B.V.Brown; individualCount: 3; sex: male; lifeStage: adult; Location: country: USA; stateProvince: California; county: San Luis Obispo; locality: Rancho El Chorro; verbatimElevation: 110 m; decimalLatitude: 35.34; decimalLongitude: -120.73; Event: samplingProtocol: Malaise trap; verbatimEventDate: 5-8.vii.2017; habitat: forest near stream; Record Level: collectionID: LACM; basisOfRecord: Preserved specimenType status:Paratype. Occurrence: recordedBy: P.H.Arnaud; individualCount: 7; sex: male; lifeStage: adult; Location: country: USA; stateProvince: California; county: San Mateo; locality: Palo Alto, Stanford University; Event: samplingProtocol: Malaise trap; verbatimEventDate: 1-15.i.1995; Record Level: institutionID: CAS, CSCA, LACM; basisOfRecord: PreservedSpecimenType status:Paratype. Occurrence: recordedBy: P.H.Arnaud; individualCount: 3; sex: male; lifeStage: adult; Location: country: USA; stateProvince: California; county: San Mateo; locality: Palo Alto, Stanford University; Event: samplingProtocol: Malaise trap; verbatimEventDate: 26-31.xii.1994; Record Level: institutionID: LACM; basisOfRecord: PreservedSpecimenSee Table Megaselia, except those similar to M.sulphurizona, by the contrasting white colour of the posterior abdominal tergites. The lighter coloured halter, stronger ventral abdominal setae , a sycamore/oak forest near a small creek, a coastal floodplain, an old field near the coast, to an inland urban backyard. Many involve at least some exposed water, but this might be simply a reflection of where we put our Malaise traps.


Histological chorioamnionitis (HCA) is one of the leading causes of spontaneous preterm birth, thus, to identify novel biomarkers for the early diagnosis of HCA is in a great need.To investigate the diagnostic value of maternal peripheral blood platelet‐to‐white blood cell ratio (PLT/WBC) and platelet (PLT) counts in HCA‐related preterm birth.A total of 400 patients with preterm birth were enrolled in this study: non‐HCA group (n = 193) and HCA group (n = 207), and 87 full‐term pregnancies were enrolled as the control. The peripheral blood of the participators was collected, and the neutrophil count, WBC count, platelet count, and levels of C‐reactive protein (CRP) and procalcitonin were recorded, and the platelet‐to‐white blood cell ratio (PLT/WBC) of the participators was calculated. Receiver operating characteristic (ROC) curve has been drawn to show the sensitivity and specificity of PLT/WBC and PLT count for the diagnosis of HCA‐related spontaneous preterm birth patients.P < 0.05) were significantly increased in HCA group compared with non‐HCA group; moreover, the area under the curve (AUC) of PLT/WBC, PLT, and CRP was 0.744 , 0.8095 , and 0.5730 , respectively.The neutrophil count, WBC count, and procalcitonin show no significant differences among the three groups, and the PLT count, PLT/WBC, and CRP (Platelet count and PLT/WBC may become a potential biomarker of HCA‐related spontaneous preterm birth. Based on previous data, the incidence rate of preterm is more than 10% of the total births, and it is also one of the leading causes of pregnancy‐related deaths worldwide .3) and maternal tachycardia (>100 bpm), there are no obvious early symptoms for HCA, as a result, to identify novel biomarkers for the early diagnosis of HCA in pregnant women is of great importance for the prevention of spontaneous preterm birth.In recent years, increasing evidence indicated that there is a strong positive correlation between the infections in the genitourinary tract and the incidence of preterm birth, which consequentially lead to the preterm birth.In recent years, studies on the serological parameters for the early diagnosis of different diseases have become an area of focus. Some previous studies demonstrated that neutrophil‐to‐lymphocyte ratio of the patients was associated with the diagnosis and outcome of different diseases, including acute cerebrovascular diseases.To our knowledge, the roles of PLT/WBC in the pathogenesis of preterm birth have not yet been discussed. In the present study, we will focus on the relationship between PLT/WBC and the chorioamnionitis‐related preterm birth. Our study may provide novel evidence for the application of PLT/WBC as potential biomarkers for the early diagnosis of HCA‐related spontaneous preterm birth.22.1A total of 400 patients with preterm birth between November 2016 and April 2017 at Fujian Provincial Maternity and Children’s Hospital, affiliated Hospital of Fujian Medical University, were enrolled in this study. The clinical information of the patients is shown in Table 2.2After delivery, tissue samples were obtained from the placenta, umbilical cord , and placental membranes of the patients. The tissue samples were then embedded into paraffin and sectioned into slices; next, hematoxylin and eosin staining has been performed for the diagnosis of chorioamnionitis. Diagnosis of HCA was determined by the infiltrations of polymorphonuclear leukocytes in the chorion‐decidua as previously described.2.3Peripheral blood specimens of the patients were collected when they first time come to the outpatient department of our hospital, and none of them received the treatment of corticosteroids, tocolytic agents, or antibiotics. The blood samples of the full‐term pregnancies were collected at the active phase of the first stage of labor. The serological analysis was performed by the clinical laboratory department of Fujian Provincial Maternity and Children’s Hospital, affiliated Hospital of Fujian Medical University. The neutrophil count, WBC count, platelet count, and levels of C‐reactive protein (CRP) and procalcitonin were recorded, and then, platelet‐to‐white blood cell ratio (PLT/WBC) of the participators was calculated.2.4P < 0.05 was considered as clinical significance.All statistical analyses were performed using the SPSS 19 software package . Data were presented as mean ± standard deviation (SD). Analysis of variance (ANOVA) has been applied for the comparison of PLT, WBC, NE, and PLT/WBC, has been applied to compare differences among different groups, and Bonferroni correction was used for multiple comparisons. The receiver operating characteristic curve (ROC) has been assessed to determine the diagnostic value of PLT count as well as PLT/WBC 33.1P > 0.05). Compared with the control group, the gestational age and birthweight of the infants were significantly lower in both non‐HCA and HCA groups (P < 0.05), and the body mass index in non‐HCA group and HCA group was markedly higher than in the control group (P < 0.05).The clinical characteristics of the participators are shown in Table 3.2P < 0.001), and the platelet count in HCA group was significantly higher than in non‐HCA group (P < 0.001). On the other hand, the PLT/WBC was significantly increased in HCA group compared with non‐HCA group (P < 0.01), and it has shown no significant differences between the control and non‐HCA group ; on the other hand, procalcitonin shows no significant difference among the three groups (P > 0.05).Moreover, the levels of inflammatory biomarkers CRP and procalcitonin among different groups were compared. As shown in Figure 3.4Finally, the receiver operating characteristic curves (ROC) have been drawn to show the sensitivity and specificity of PLT/WBC, PLT count, and CRP to distinguish HCA patients from non‐HCA patients. As shown in Figure 4In the present study, the roles of PLT/WBC and PLT counts as potential early diagnostic markers for HCA have been discussed. We proved that both PLT/WBC and PLT count were significantly upregulated in patients with HCA, and either PLT/WBC or PLT count was a sensitive biomarker that can distinguish HCA patients from non‐HCA patients with spontaneous preterm birth. Our results have proposed the potential diagnostic value of PLT/WBC and PLT count for the early diagnosis of HCA‐related spontaneous preterm birth.P > 0.05). On the other hand, the activation of platelets can be found in the pathophysiological changes in many biological processes, that is, infection, inflammation, and tumorigenesis. In the case of inflammation, platelets were rapidly recruited and participated in the pathogenesis of inflammation by secreting cytokines, chemokines, and other inflammatory mediatorsP < 0.001), and the platelet count in HCA group was significantly higher than in non‐HCA group (P < 0.001); moreover, receiver operating characteristic curve has been drawn to evaluate the diagnostic value of PLT/WBC to distinguish HCA patients from non‐HCA patients. The AUC of PLT count is 0.8095, suggesting that PLT count is a sensitive indicator of HCA among patients with preterm birth. Interestingly, we also observed that CRP, a well‐known inflammatory biomarker, was also elevated in HCA patients, and the AUC of CRP to distinguish HCA patients from non‐HCA patients was 0.5730, indicating that CRP is a not a sensitive biomarker to distinguish HCA patients from non‐HCA patients. Taken together, these results suggested that high maternal peripheral blood level of platelet may indicate the risk of HCA‐related spontaneous preterm birth.Intrauterine infection is one of the most common obstetric complications that is induced by chronic or acute inflammation of the membranes and chorion of the placenta, leading to an increased risk of preterm birth. It was estimated that about 40% of cases of preterm birth have been correlated with intrauterine infection.P < 0.01); next, results of ROC analysis indicated that the AUC of PLT/WBC was 0.744, suggesting that PLT/WBC is a sensitive biomarker for the diagnosis of HCA. The mechanistic relationship between PLT/WBC ratio and infection remains unclear. One possibility is that the PLT/WBC ratio is an indicator for a patient’s baseline health status. PLT/WBC ratio is also likely an indicator of metabolic syndrome, a constellation of physiological and biochemical abnormalities, resulting in inappropriate activation of inflammatory pathways. Nevertheless, we first reported that PLT/WBC ratio may serve as a biomarker for the early diagnosis of HCA.It has been proved in many previous studies that the ratio of immune cells  in the peripheral blood may serve as inexpensive and reproducible biomarkers. Increasing evidences indicated that peripheral blood PLT/WBC ratio may serve as a biomarker for the early diagnosis, evaluation of the therapeutic efficacy, and prognosis of many diseases.Our studies have limitations. First, the results should be verified with larger sample size in future studies; second, this study was based on Chinese Han population, so whether PLT/WBC and PLT have diagnostic value for HCA in other races still needs to be validated.In conclusion, we reported for the first time that PLT/WBC and PLT count were significantly upregulated in patients with HCA‐related spontaneous preterm birth, and our results have provided novel evidence that PLT/WBC and PLT count were sensitive biomarkers for the early diagnosis of HCA‐related spontaneous preterm birth.


Despite the widespread clinical evidences for the association of Mg2+ deficiency (MgD) and type 2 diabetes mellitus (T2D), molecular mechanisms by which Mg2+ contributes to insulin resistance (IR) are still under discussion. Mg2+ regulates electrical activity and insulin secretion in pancreatic beta-cells. Intracellular Mg2+ concentrations are critical for the phosphorylation of the insulin receptor and other downstream signal kinases of the target cells. Low Mg2+ levels result in a defective tyrosine kinase activity, post-receptor impairment in insulin action, altered cellular glucose transport, and decreased cellular glucose utilization, which promotes peripheral IR in T2D. MgD triggers chronic systemic inflammation that also potentiates IR. People with T2D may end up in a vicious circle in which MgD increases IR and IR causes MgD, that requires periodic monitoring of serum Mg2+ levels.Magnesium (Mg Insulin resistance (IR) is associated with an impaired biological response to insulin stimulation of key target tissues, particularly liver, muscle, and adipose tissue. IR impacts glucose utilization, resulting in a compensatory increase in beta-cell insulin production and hyperinsulinemia . Progres2+) is the fourth most common mineral in the human body, after calcium (Ca2+), potassium (K+), and sodium (Na+), and the second most abundant intracellular cation after K+ . In. In2+ oron MgATP . In the d F1,6BP . Glycoged F1,6BP . GSK3 isd F1,6BP . Mg2+ diith Mg2+ . Mg2+ is enzymes ,51. Mg2+1-ATPase .2+ concentrations are determined by various Mg2+ channels and transporters. Of these, transient receptor potential melastatin type 6 and 7 (TRPM6 and TRPM7) ion channels, solute carrier family 41 member 1 (SLC41A1), and Mg2+ transporter 1 (MagT1) play a major role. Several groups of researchers have investigated the association between genetic variations in these Mg2+ transporters and risk for T2D. Until now, single nucleotide polymorphisms have been found in the TRPM6 and SLC41A1 genes, which are associated with an increased risk for T2D [2+ ions. Low Mg2+ intake and imbalance in intracellular Mg2+ concentrations may interact with KATP ion channel variants in affecting T2D risk [Intracellular Mg for T2D . Patient for T2D ,52,53. AT2D risk   for Mgor women . In the c region . Dietary5 mg/day . Dietarypulation  and in mpulation . This ispulation . Refininly needs . Water it extent . In drin100 mg/L . Therefover time . Inadequtes risk . Van Damk of T2D .2+ homeostasis is governed by reabsorption of Mg2+ from primary urine in the kidney. Of about 2,400 mg Mg2+ that is ultra-filtrated daily, 95–99% is reabsorbed by the nephrons. The causes of Mg2+ loss through the kidneys are elucidated in detail during the recent years and may involve various damage of the transport systems located in the thick ascending limb (TAL) of Henle’s loop and distal convoluted tubules [2+ waste are Gitelman syndrome, Bartter syndrome, familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), autosomal-dominant hypocalcemia with hypercalciuria (ADHH), isolated dominant hypomagnesemia (IDH) with hypocalciuria, isolated recessive hypomagnesemia (IRH) with normocalcemia, and hypomagnesemia with secondary hypocalcemia (HSH) [2+ waste. Mg2+ uptake in the TAL of Henle may also have a role, since hyperinsulinemia and IR may lead to a decreased Mg2+ uptake and an increase Mg2+ excretion. Hyperglycemia also leads to increased urinary Mg2+ waste, contributing to Mg2+ depletion. Plasma Mg2+ levels were found inversely correlated with the urinary Mg2+ excretion rate and with fasting blood glucose values, suggesting that the tubular reabsorption of Mg2+ is decreased in the presence of severe hyperglycemia [2+ excretion through the inhibition of the electrical gradient necessary for Mg2+ reabsorption in the TAL. Long-term thiazide diuretic therapy also may cause MgD, through enhanced Mg2+ excretion and, specifically, reduced renal expression levels of the epithelial Mg2+ channel TRPM6. Many nephrotoxic drugs, including cisplatin, cyclosporine, and tacrolimus, can produce urinary Mg2+ waste by a variety of mechanisms, some of which are still unknown [Mg tubules ,12. Inheia (HSH) ,66. Receglycemia . Several2+ absorption occurs predominantly in the small intestine via a paracellular pathway and smaller amounts are absorbed into the colon, mainly via a transcellular pathway. Paracellular Mg2+ absorption occurs through simple diffusion and involves the transport of Mg2+ through small spaces between epithelial cells and depends on specialized transmembrane proteins, claudins, which control ion permeability. The transcellular pathway of Mg2+ absorption depends on the TRPM6 and TRPM7 transporters. Various factors influence the intestinal uptake of Mg2+ and are of substantial importance for the supply of this mineral [2+ absorption is a common underlying basis for hypomagnesemia, especially when the small bowel is involved, due to disorders associated with malabsorption, chronic diarrhea, or steatorrhea, or as a result of bypass surgery on the small intestine. As there is some Mg2+ absorption in the colon, patients with ileostomies can develop hypomagnesemia [2+ absorption may be impaired in a number of diseases, e.g., Crohn’s disease, ulcerative colitis, coeliac disease, short bowel syndrome, and Whipple’s disease [2+ when the intraluminal intestinal concentration of Mg2+ is low [2+. Hypomagnesemia is a recognized side-effect of PPIs [Intestinal Mg mineral . Impairegnesemia . Mg2+ ab disease ,57. Gene+ is low . Proton  of PPIs . Use of  of PPIs . This ma2+ supplementation in diabetic subjects have been found in a number of clinical studies. They demonstrate that oral administration of Mg2+ reduces IR and improves insulin sensitivity in T2D patients, as well as in overweight nondiabetic subjects  and increases to 420 mg/day for males 31–70 years of age and older. For females, the RDA ranges from 240 mg/day (9–13 years of age) to 360 mg/day for females 14–18 years of age. The RDA for females 31–70 years of age and older is 320 mg/day. Many nutritional experts feel the ideal intake for Mg2+ should be based on the body weight  [2+ salts, such as Mg2+ citrate, gluconate, orotate, or aspartate, are recommended in the treatment of MgD because of their high bioavailability [2+ salts may be the risk of developing hypoglycemia by increasing the intestinal absorption rate of SUs. Therefore, SUs should be given at least 1 h before Mg2+ intake [These data indicate that special attention should be given to the risk groups, in particular individuals with MetS and T2D, in which serum Mg kg/day) . Organiclability ,78. A po+ intake .2+ on a number of mechanisms related to IR, consuming a healthy Mg2+-rich diet should be encouraged for individuals with MetS and T2D. There is consensus on the benefits of certain named dietary patterns, such as the Mediterranean diet and DASH (Dietary Approaches to Stop Hypertension) diet for prevention and management of T2D [2+, dietary fiber, antioxidants, and polyphenolic compounds [2+, K+, Ca2+, dietary fiber, and protein and smaller amounts of total and saturated fat and cholesterol than the typical diet [Considering the numerous positive effects of Mgt of T2D . The Medompounds . The DASompounds . The DAScal diet .2+ concentrations within the reference range is essential for normal insulin secretion and activity, as well as for the optimal functioning of many enzymes of glucose and energy metabolism. MgD may be associated with beta-cell dysfunction, IR, reduced glucose tolerance, and ultimately, clinical manifestations of T2D. Oral Mg2+ supplementation and appropriate dietary patterns improve insulin sensitivity and metabolic control in patients with T2D, suggesting that Mg2+ is an important factor in the etiology and management of this widespread socially significant disease.Maintaining serum Mg


Dear Editor,We read, with great interest, the article by Brockfeld et al . evaluatDavis et al.  exploredOn the contrary, access to video lectures may breed complacency with regards to managing the academic workload. The availability of video lectures relies on the student’s impetus to get through the material in their own time. A lack of motivation to work at a sufficient pace may result in the accumulation of an insurmountable workload before impending exams. Therefore, the benefit of video lectures is likely to be heavily influenced by the attitudes of the individual. This was demonstrated by Howland and Moore  who explLive lectures may provide a more structured learning schedule and promote sustainable management of a student’s workload as the rate of delivery of information is controlled by the medical school. The balance between the use of video and live lectures could be shifted according to the maturity of the students. It is, perhaps, likely that younger students would benefit more from live lectures due to inexperience with managing their workload independently. Older students, on the other hand, may prefer more independence and control of their workload as they must often juggle hospital placements and lectures. Furthermore, the type of information being delivered should also influence this balance. Some topics (e.g. biochemistry) are predominantly delivered via didactic lecture-based transfer of information, which is achieved effectively by video lectures . On the To conclude, live lectures promote a sustainable work rate and encourage engagement whilst video lectures are a convenient revision tool allowing students to work at their own pace. As such, video lectures should be viewed as a supplement to live lectures rather than a replacement. To optimise the benefit to students, medical schools should also shift the emphasis on either video or live lectures based on the maturity of the students and the type of information being delivered.


We aimed to compare VLS measurements with a validated quantitative microvascular oxygen tension . Thereafter, the influence of bile was assessed by comparing VLS measurements in the presence of bile and without bile. Finally, simultaneous VLS and μPO2 measurements were performed from the moment a lethal dose potassium chloride intravenously was injected.Simultaneous VLS measurements and μPO2 values that increased with increasing FiO2, VLS values decreased. Both measurements correlated poorly with R2 = 0.39, intercept 18.5, slope 0.41 and a bias of − 16%. Furthermore, the presence of bile influenced VLS values significantly (median (IQR)) before bile application 57.5% (54.8–59.0%) versus median with bile mixture of the stomach 73.5% (66.8–85.8), p = < 2.2 * 10−16; median with bile mixture of small bowel 47.6% (41.8–50.8) versus median after bile removal 57.0% (54.7–58.6%), p = < 2.2 * 10−16). Finally, the VLS mucosal oxygen saturation values did not decrease towards a value of 0 in the first 25 min of asystole in contrast to the μPO2 values.In contrast to μPOThese results suggest that VLS measures the mixed venous oxygen saturation rather than mucosal capillary hemoglobin oxygen saturation. Further research is needed to establish if the mixed venous compartment is optimal to assess gastrointestinal ischemia. Visible light spectroscopy (VLS) is a technique used to measure the mucosal capillary hemoglobin oxygen saturation based on reflectance spectrophotometry . The mucVLS is used in clinical practice in the work-up of the diagnosis of chronic mesenteric ischemia (CMI). CMI is defined as ischemic symptoms caused by insufficient blood supply to the gastrointestinal (GI) tract . The maiThe diagnosis of CMI is a clinical challenge because of the diverse presentation of CMI. Symptoms overlap largely with many other disorders and the high prevalence of asymptomatic mesenteric artery stenosis in the general population of 3–29% , 9) due % , 9 dueIn the absence of one specific test for the diagnosis of CMI , the dia2) measurement technique . The formula was derived from 213 data point at pH 7.4 and 37° with an excellent fit.Statistical analysis was performed with R Statistics software (v3.2.4). Normal distribution was assessed visually and with the Shapiro–Wilk normality test. Normal distributed data is presented as mean ± standard deviation (SD) and abnormally distributed data is presented as median with interquartile range (IQR). A linear regression model was used for the FiOwas used : (%/100)2 is measured on demand. To equally compare the two measurements the mucosal oxygen saturation was averaged over same period as one μPO2 was done. Thereafter these results were visualized with linear regression and with a Bland–Altman comparison plot [To compare the saturation, the difference in measurement frequency had to be overcome. The mucosal oxygen saturation has a fixed measurement interval whereas the μPOson plot .2. Because VLS measures every second, a symmetrical moving average of 20 samples was taken to smooth the data, for example the eleventh sample is an average of sample [The Wilcoxon signed-rank test was used to compare the measurement before, with and after application of bile. A two-tailed p value of < 0.05 was considered significant. After the potassium chloride injection mucosal oxygen saturation measurements were compared with μPOf sample –21.All 5 animals were in good clinical condition before the start of the experiment.2 levels different values of FiO2 in 5 animals were measured. The mucosal oxygen saturation decreased with increasing FiO2 in contrast to the μPO2 values that increased with increasing FiO2. The spread of the mucosal oxygen saturation levels and the FiO2 levels was large, shown in Fig. The mucosal oxygen saturation levels versus the μPO2 saturation. There is a poor linear correlation with an r2 = 0.39, an interception of 18.5% and a slope of 0.41. In the Bland–Altman plot . When the bile mixture from the small bowel was applied, the mucosal oxygen saturation was significantly lower with a median (IQR) 47.6% (41.8–50.8), p = < 2.2 * 10−16 compared to mucosal oxygen saturation measurements with bile mixture form the stomach and the mucosal oxygen saturation increased significantly after the bile mixtures had been removed (p = < 2.2 * 10−16).Figure 2 measurements during the minimally first 25 min of asystole in 5 animals are shown in Fig. 2 measurements decreased towards a value of 0. The mucosal oxygen saturation measured with VLS decreased and increased variably during the measurement period and the mucosal oxygen saturation never reached a stable state around 0%.The mucosal oxygen saturation measurements and μPONo adverse events occurred during the 5 porcine experiments.2 measurements. This study showed that the mucosal oxygen saturation values decreased with increasing FiO2 in contrast to the μPO2 values that increased with increasing FiO2 with a large spread of the measured mucosal oxygen saturation levels and FiO2 levels and a poor linear correlation. Furthermore, a significant influence of bile on the mucosal oxygen saturation values was shown. Finally, this study showed that the mucosal oxygen saturation values, in contrast to the μPO2 values, did not decrease towards a value of 0 in the first 25 min of asystole.In this study we validated mucosal oxygen saturation measurements by comparing VLS with calibrated μPO2 is remarkable. Mucosal oxygen saturations measured with VLS are expected to increase with increasing FiO2 if VLS measures the capillary oxygen saturation level. However, VLS measures not only arterial saturation but also a large venous compartment. If a large mixed venous saturation determines the overall saturation value the influence of FiO2 is expected to be minimal. Potentially due to hyperoxic vasoconstriction the actual venous saturation can decrease more compared to normoxic situations. The high FiO2 values will be measured by the μPO2. Furthermore, the measured values, both VLS as μPO2 values, have a great spread. Possibly, the oxygen tension was very variable in the gastrointestinal vessels as intestinal ischemia is also patchy and heterogenic distributed [The found inverse relationship of the mucosal oxygen saturation measurements by VLS with FiOtributed . During Significant influence of bile on the mucosal oxygen saturation values measured with VLS was confirmed. Therefore it is advised and mentioned in the prescription to remove any bile remnants before the start of the VLS measurements. The bile has its own absorption spectrum of light. It also absorbs light in the same wavelengths as oxyhemoglobin and deoxyhemoglobin , and infThe idea that VLS measures mixed venous oxygen saturation is further confirmed by the fact that VLS measured still a reasonable oxygen saturation 25 min after asystole. The saturation in the capillaries is decreased towards zero over time due to diffusion of oxygen towards the still oxygen consuming cells. However, in the venous compartment the oxygen will desaturate slowly by the large buffer capacity. Therefore, the oxygen saturation will not decrease towards zero immediately after asystole. Dips in oxygen saturation are seen in the mixed venous compartment measured by VLS as shown in Fig. 2, which is an exaggerated model of instant ischemia.VLS is a powerful technique to measure oxygen saturation at a microvascular level. In the microvasculature oxyhemoglobin/deoxyhemoglobin is proportional mainly located in the venous compartment of the microvasculature. Therefore the saturation measured by the VLS is mainly represented by the venous compartment. For detection of an oxygen transport problem that results in ischemia, the microvascular arterial saturation is of importance, a part that is underexposed by VLS. This is endorsed by the fact that after a lethal potassium chloride the saturation does not drop in comparison to μPO2 of the mucosa of the small intestine of a pig, the abdomen had to be opened to open the small intestinal loop. The mucosa of this small intestinal loop was exposed to room air and room temperature. This will result in oxygen diffusion into the tissue and rapid decrease in temperature for the exposed tissue. Furthermore, the abdominal anatomy of a pig is different from the human abdominal anatomy. The GI tract of a pig is monogastric like the human GI tract, however the colon lies in a spiral. The mesenteric vascularization in humans consists of individual variable, mesenteric vessel formations with arcades, lateral branches and anastomoses in the bowel wall [This study has some limitations. First, the experiments performed in this study were designed to enable generalizability in humans. However, to enable stable oxygen saturation measurements with VLS and μPOwel wall . The meswel wall .This study showed that VLS measures the mixed venous hemoglobin oxygen saturation and not the mucosal capillary hemoglobin oxygen saturation. The presence of bile significantly influences the oxygen saturation levels measured with VLS. VLS is currently used in clinical practice in the clinical work-up of CMI. Further research is needed to establish if the mixed venous compartment is optimal for mucosal hemoglobin saturation measurements to assess GI ischemia.


Fusarium metabolite, was recently found to inhibit the corresponding detoxification reaction in plants, namely DON-glucoside formation, raising the question whether CUL might affect also the mammalian counterpart. Using cell-free conditions, CUL when present equimolar (67 µM) or in fivefold excess, suppressed DON glucuronidation by human liver microsomes, reducing the formation of DON-15-glucuronide by 15 and 50%, and DON-3-glucuronide by 30 and 50%, respectively. Substantial inhibitory effects on DON glucuronidation up to 100% were found using the human recombinant uridine 5′-diphospho-glucuronosyltransferases (UGT) 2B4 and 2B7, applying a tenfold excess of CUL (100 µM). In addition, we observed the formation of a novel metabolite of CUL, CUL-11-glucuronide, identified for the first time in vitro as well as in vivo in piglet and human urine samples. Despite the observed potency of CUL to inhibit glucuronidation, no significant synergistic toxicity on cell viability was observed in combinations of CUL (0.1–100 µM) and DON (0.01–10 µM) in HT-29 and HepG2 cells, presumably reflecting the limited capacity of the tested cell lines for DON glucuronidation. However, in humans, glucuronidation is known to represent the main detoxification pathway for DON. The present results, including the identification of CUL-11-glucuronide in urine samples of piglets and humans, underline the necessity of further studies on the relevance of CUL as a potentially co-occurring modulator of DON toxicokinetics in vivo.Glucuronidation is a major phase II conjugation pathway in mammals, playing an important role in the detoxification and biotransformation of xenobiotics including mycotoxins such as deoxynivalenol (DON). Culmorin (CUL), a potentially co-occurring The online version of this article (10.1007/s00204-019-02459-w) contains supplementary material, which is available to authorized users. Fusarium, Aspergillus, Penicillium and Alternaria. As toxigenic molds frequently contaminate agricultural crops pre- or post-harvest, mycotoxins may enter the food and feed chains, posing a potential risk to both human and animal health. Recent multi-mycotoxin surveys as well as human biomonitoring studies point at co-occurrence of various mycotoxins , has been investigated in several studies   N-acetylglucosamine were obtained from Sigma Aldrich . For mass spectrometric measurements, methanol (MeOH), acetonitrile (ACN), acetic acid and water of LC–MS grade were purchased from Sigma . A multi-component standard including DON and its metabolites  was prepared according to Warth et al. , (2) CUL/DON , (3) CUL/DON . The concentration of DON in all cases was 20 mg/L (67 µM), while the molar excess of the co-substrate UDPGA was at least 20-fold. After stopping the reactions, samples were diluted 1:10 with 20% aqueous methanol and the formed glucuronides were determined by LC–MS/MS.The potential inhibitory effect of CUL on DON glucuronidation was analyzed using liver microsomes from rats or humans. In principle, the assays were performed as described previously for DON alone  or CUL (100 µM) was added and the reaction was initiated by the addition of UDPGA (2.5 mM). After further incubation for 1 h at 37 °C, reactions were terminated by addition of 200 µL ACN. Samples were then chilled at − 20 °C for 10 min and centrifuged at 14,000×g for 5 min. Then, 200 µL of the supernatant were evaporated to dryness using a Labconco Centrivap Benchtop Vacuum Concentrator  and reconstituted in 100 µL water/ACN  for LC–MS/MS analysis. Glucuronidation experiments with the recombinant UGTs were carried out in four independent experiments.Furthermore, the effect of CUL on the glucuronidation of DON was tested using two different UGT isoforms  according to Maul et al. . Human UAll procedures related to the animal experiment were performed according to Austrian law and following the European Guidelines for the Care and Use of Animals for Research Purpose  were not detected.Six crossbred piglets  were obtained from a local swine producer. Piglets were housed individually in metabolic cages, had free access to water and were allowed to acclimatize for 5 days. Feed was provided twice daily and withdrawn after 30 min to monitor feed intake. Prior to the start of the experiment, the feed was tested for mycotoxin contamination by LC–MS/MS  and CUL (150 µg/kg body weight) by gavage on day 1 and 7. To this end, respective solutions were orally administered via polyvinyl chloride catheters. Urine and feces of individual piglets were collected 0–8 h, 8–24 h and 24–28 h after each of the treatments. Samples were stored at − 20 °C until analysis.g for 10 min. Measurement of liver microsome samples and pig urine samples was carried out on a 1290 Infinity series UHPLC system  coupled to a 6500+ QTrap mass spectrometer equipped with an IonDrive TurboV source . Analytes were separated in gradient elution mode on a Kinetex C18 column . Mobile phase A was water/acetic acid , mobile phase B was composed of ACN/acetic acid . The following linear gradient was used: 0.0–0.5 min: 5% B, 0.5–7.0 min: linear increase to 15% B, 7.0–14.0 min: linear increase to 90% B, 14.1–16.0 min: 100% B, 16.1–19.0 min: re-equilibration at 5% B. The flow rate was 250 µL/min, the column temperature was 30 °C and the injection volume was 3 µL. Mass spectrometric detection was performed in negative mode after electrospray ionization. The source parameters were: source temperature 400 °C, ion spray voltage − 4500 V, curtain gas 35 psi, ion source gas 1 60 psi and ion source gas 2 40 psi. Selected reaction monitoring mode was chosen as scan type and the following transitions were used: CUL-11-GlcAc quantifier m/z 413.5 → m/z 113.1 , CUL-11-GlcAc qualifier_1 m/z 413.5 → m/z 175.1 , CUL-11-GlcAc qualifier_2 m/z 413.5 →  m/z 219.2 ; CUL quantifier m/z 297.4 → m/z 59.0 . Analyst software version 1.6.3 (SCIEX) was used for instrument control and data analysis. CUL-11-GlcAc eluted at 11.40 min, CUL at 12.89 min. In urine diluted to 0.2 mM creatinine, matrix effects of CUL-11-GlcAc and CUL were 96 and 95%, respectively. The limits of detection  for CUL-11-GlcAc and CUL in pure solvent standard solution were 0.1 ng/mL and 2 ng/mL, respectively. In urine diluted to 0.2 mM creatinine, the LOD of CUL-11-GlcAc was 0.8 ng/mL and the LOD of CUL was 2.5 ng/mL. Limits of quantification  were by the factor 3.3 higher than LODs.Pig urine samples were diluted to 0.2 mM creatinine with MeOH/water  and centrifuged at 14,350×m/z 60–900) with a resolving power of 120.000 full width at half maximum and automatic gain control setting of 3 × 106 with a maximum injection time of 100 ms. The LOD for DON-3-GlcA and DON-15-GlcA (peak area 5000 a.u.) was estimated based on the smallest peak detected within the respective measurement sequence. For data evaluation, Xcalibur™  and TraceFinder™  software were used. The general status of the instrument was regularly checked by the measurement of blank and quality control samples before and after the sequence.All samples obtained from the glucuronidation assay using the recombinant enzymes UGT 2B4 and UGT 2B7 and also the human urine sample were measured by LC–HRMS applying the following method: after reconstitution, the samples were analyzed by an LC–HRMS method using a UHPLC Vanquish system hyphenated with a Q Exactive HF high-resolution mass spectrometer equipped with a heated electrospray ionization (hESI) source . Chromatographic separation was achieved on a reversed-phase Acquity UPLC HSS T3 column  protected by a HSS T3 VanGuard pre-column at 40 °C. At a constant flow rate of 0.4 mL/min, a linear gradient was employed with water (A) and ACN (B), both containing 0.1% acetic acid. After an initial hold time of 2 min at 10% B, the gradient was raised to 50% B at minute 6 and 100% B at minute 7. After 2 min at 100% B, the column was re-equilibrated at 10% B for 1 min. A sample volume of 2 μL was injected by the autosampler which was maintained at 4 °C. The following settings were used: negative ionization mode, capillary temperature, 325 °C; vaporizer temperature, 400 °C; sheath gas, 60 arbitrary units (a.u.); auxiliary gas, 20 a.u.; sweep gas, 3 a.u.; capillary voltage, 3 kV. The instrument was operated in profile mode  LC–MS grade water and 1% (v/v) DMSO. Measurements of combinatory effects were always performed in parallel to the single substance on the same 96-well plate, to maximize comparability of the data and to allow an accurate calculation of the combinatory effects.The human colorectal adenocarcinoma cell line HT-29 was purchased from the German Collection of Microorganisms and Cell Cultures , the human liver hepatocellular carcinoma cells HepG2 and the human colorectal adenocarcinoma cell line Caco-2 from the American Type Culture Collection . For HT-29 and Caco-2 cell cultivation Dulbecco’s Modified Eagle’s Medium and for HepG2 cells RPMI 1640 medium was used. All three basal media formulations were supplemented with 10% (v/v) heat-inactivated fetal calf serum and 1% (v/v) penicillin/streptomycin (50 U/mL). Caco-2 culture medium was additionally supplemented with 1% potassium pyruvate and 0.01 mg/mL insulin–transferrin–selenium. Culture media and supplements were purchased from GIBCO Invitrogen . For cell cultivation and treatments, humidified incubators at 37 °C and 5% CO® Mini Kits  and reversed transcribed into complementary DNA (cDNA) by QuantiTect® Reverse Transcription Kit (Qiagen) according to the supplier’s protocols. Amplification of the cDNA samples in the presence of gene-specific primers  and QuantiTect® SYBR Green Master Mix (Qiagen) was performed in technical duplicates using a StepOnePlus™ System . The following primer assays were used: β-actin ; glyceraldehyde 3-phosphate dehydrogenase ; UGT 2B4 ; UGT 2B7 . A universal PCR protocol was applied including 15-min enzyme activation at 95 °C, 40 cycles of 15 s at 94 °C, 30 s at 55 °C and 30 s at 72 °C. StepOnePlus® software  was used for fluorescence signal quantification and further data analysis. Of each tested sample, at least three independent experiments were performed. Presented transcript data were normalized to the mean of transcript levels of endogenous control genes  applying if possible and necessary the ΔΔCt-method .Gene transcription of UGT 2B4 and UGT 2B7 in three different human cancer cell lines  and the potential impact of CUL and DON on UGT transcription levels was analyzed by quantitative real-time PCR (qPCR). Cells were seeded in 24-well plates  and allowed to grow for 48 h. After respective incubations, total RNA was extracted using RNeasyTo determine combinatory effects of CUL with DON on cell viability, the sulforhodamine B (SRB) assay according to Skehan et al.  was appl50 value and, therefore, a measurable effect of at least 50% is a prerequisite for the application of this model. Since in some of our experimental setups this criterion was not fulfilled, the model of independent joint action (IJA) was chosen for the analysis of combinatory effects of CUL and its co-contaminant DON , defined as LOD. For statistical analysis and data visualization, Origin 2018 software  was used.Combinatorial interactions of two or more substances are present if the experimentally determined effect differs from the “additive effect” of the two or more single compounds Chou . To asseDON Webb . ApplyinThe ability of CUL to interfere with DON glucuronidation was assessed using two commercially available liver microsome preparations from human and rats. As expected, the main metabolite of DON incubated with liver microsomes from rats was DON-3-GlcA (data not shown). No significant concentration differences of DON-3-GlcA in the DON, CUL/DON (1:1) and CUL/DON (1:5) groups were seen. Thus, it seems that CUL has no modulatory effect on the glucuronidation activity of rat liver microsomes. Interestingly, the picture was quite different using human liver microsomes Fig. a, b. OnlIn case of human recombinant UGTs, both UGT 2B isoforms tested (2B4 and 2B7), accepted DON as a substrate, leading to the formation of one glucuronide each  levels of the two UGT isoforms responsible for DON glucuronidation in three different cancer cell lines were investigated by qRT-PCR and are depicted relative to UGT 2B4 mRNA expression in HepG2 cells Fig. . TranscrThe human colon cancer cell line HT-29, known to effectively glucuronidate other mycotoxins such as alternariol and alternariol methyl ether  did not cause any statistically significant cytotoxic effect in the SRB assay after 24-h incubation Fig. a, b. SinTo assess combinatory effects of CUL on DON-induced cytotoxicity after longer incubation times, HT-29 cells were treated for 48 h. Nevertheless, even cell treatments for 48 h did not cause significant cytotoxic effects of CUL, nor combinatory effects of CUL and DON Fig. c. Thus, Urine samples of the piglet trial were diluted to the same concentration of creatinine (0.2 mM) and concentrations of CUL and CUL-11-GlcA were determined by LC–MS/MS. CUL-11-GlcA eluted at 11.40 min, CUL at 12.89 min and a potential further CUL-GlcA at 4.27 min. In urine diluted to 0.2 mM creatinine, matrix effects of CUL-11-GlcA and CUL were 96 and 95%, respectively. The LODs  for CUL-11-GlcA and CUL in pure solvent standard solution were 0.1 ng/mL and 2 ng/mL, respectively. In urine diluted to 0.2 mM creatinine, the LOD of CUL-11-GlcA was 0.8 ng/mL and the LOD of CUL was 2.5 ng/mL. Limits of quantification  were by the factor 3.3 higher than LODs. Figure As with the recombinant UGT enzymes and (human and rat) liver microsomes, CUL was also readily glucuronidated in vivo to CUL-11-GlcA by pigs. In measured urine samples from four animals, the concentration of CUL-11-GlcA was higher than of CUL . In urine samples of two animals, no CUL-11-GlcA was detected. One likely explanation for the latter is that insufficient separation of urine and feces in the metabolic cages led to hydrolysis of the formed glucuronides by gut microbes during the sampling period.CUL-11-GlcA was further detected in a human urine sample obtained from pregnant women in Croatia  were tested for their UGT transcription levels Fig. . Even th Nakajin  reported Nakajin .1, β-naphthoflavone, ethanol,etc.) may increase UGT levels and respective glucuronidation activities via different induction pathways  and by Dowd et al. . Thus, as also transcription levels of UGT 2B4 were about tenfold lower than levels reported in liver tissue Below is the link to the electronic supplementary material.


This article comments on:Seibert T, Abel C, Wahl V. 2020. Flowering time and the identification of floral marker genes in Solanum tuberosum ssp. andigena. Journal of Experimental Botany 71, 986–996. It’s dark green and has a stalk attached. Anyone know what it might be?’ This was a question submitted by a gardener to an online discussion forum , and it Solanum tuberosum) originates from Central and South America, of course, and the first plants brought to Europe in the 16th century were of the andigena subspecies (Solanum tuberosum ssp. andigena) from the Andean Altiplano (high plain) of west-central South America. As such, they were adapted to conditions in which day length did not change much through the year and, when grown in the higher latitudes of Europe, would not tuberize until the autumn, when day length had reduced to ~12 h. This made the potato difficult to cultivate in higher latitudes, although by the early 19th century it was already a staple in countries such as Ireland where the mild climate was conducive. Efforts by breeders to develop varieties that would tuberize earlier in the season under European and North American conditions were accelerated by the incorporation of genotypes from Chile in the mid-19th century, including the wonderfully named Rough Purple Chili potato (tuberosum (Solanum tuberosum ssp. tuberosum) and enabled the development of early-maturing varieties such as Garnet Chili, Early Rose, and Burbank, the last being selected by the most famous plant breeder of the time, Luther Burbank (1849–1926). These varieties were produced by sexual crossing, but potato is an outbreeder, with most genotypes strictly self-incompatible, so once a suitable genotype had been selected from a cross it would be bulked up vegetatively, and genetically identical ‘seed’ potatoes would be sold to farmers for cultivation. Luther Burbank subsequently selected and multiplied a mutant of Burbank that was originally marketed in 1902 as Netted Gem : inbred lines would be crossed to exploit the phenomenon of heterosis, in which hybrid offspring would outperform either parent due to the combination of alleles they inherited, with the effects of ‘poor’ alleles in each parent being reduced in the hybrid offspring. The technique has been widely used in plant breeding for many crop species for decades, but in potato it has been made impossible by self-incompatibility and inbreeding depression. That may be about to change, because, in 2011, it was reported that the company Solynta, a Wageningen University spin-off, had overcome these limitations. This was achieved by introducing a self-compatibility restorer gene, Sli, from the wild species Solanum chacoense into diploid germplasm from the Wageningen University potato breeding programme and developing inbred, self-compatible genotypes , and the model fruit plant, tomato (Solanum lycopersicum)  that are eaten, flowering control has a direct effect on yield and has been studied extensively , which encodes a transcription factor, was specifically expressed in the inflorescence meristem, as it is in tomato  and the bZIP transcription factor gene FD suggested that these genes might have similar roles to their Arabidopsis counterparts in regulating flower development.The team also identified marker genes for the floral transition and flower organ development, drawing on knowledge generated in the model plant, Arabidopsis (ersicum) . Tomato,The protocols developed in the study and the results obtained will aid other researchers working on potato flowering and, although they are still in development, if hybrid potatoes fulfil their promise we are likely to see a lot more research on the genetic control of potato flowering in the future.


Objective: Determine the prevalence of Dop, a system of labor payment via alcoholic beverages, in a South African province, and its influence on maternal drinking and fetal alcohol spectrum disorders (FASD). Methods: Data from studies of FASD epidemiology were analyzed. Results: Forty-two percent to 67% of mothers reported drinking. In 1999, 5% of women reported Dop allocations in their lifetime: 14% of mothers of FASD children and 1% of controls. In 2010, 1.1% of mothers reported lifetime Dop: 1.6% of FASD mothers and 0.7% of controls. Commercial alcohol sales have replaced the Dop system. Total FASD rates remained high in rural areas in 2010 and rose in urban settings. Urban rates of total FASD surpassed rural area rates in 2010. Correlation analysis did not reveal a strong or significant, direct relationship between Dop experience and heavy drinking , or the diagnosis of FASD in children . Conclusion: Dop, as a systematic practice, is dead and does not have a direct influence on alcohol availability, heavy maternal drinking, or the probability of an FASD diagnosis. Nevertheless, today’s problematic drinking patterns were heavily influenced (shaped) by Dop and have negatively impacted the prevalence and severity of FASD.  In the Western Cape Province (WCP) of the Republic of South Africa (SA), many people are involved in agriculture, especially viticulture, the growing of grapes, and the production of wine. SA is known globally for producing, selling, and exporting popular and fine wines of many varieties. Many workers also live or have lived on the farms. Over a span of at least two centuries, and under the system of apartheid, many farm and vineyard workers in the WCP were provided with wine as partial payment for their labor ,2,3. ThiThe five communities studied to date in SA have the highest documented general-population rates of FASD in the world . From boCommunity studies on the epidemiology of FASD that were carried out by other researchers in other communities of SA ,18,19,20The populations studied here have changed substantially since the early 1990’s. In fact, some farms in the WCP have been transformed greatly and have instituted improved working conditions, worker compensation, and overall benefits . Our resData for this exploration originated from seven different population-based samples, collected between 1997 and 2011, to define the prevalence and detailed characteristics of FASD in a general population sample in the WCP. These studies were active case ascertainment (ACA) studies of FASD clinical cases found among children in community public schools. Interviews were also conducted, in person, with the mothers of the study children ,26,27,28A three-tier system was used to screen and provide diagnoses for all children in first-grade classes in community schools who had consented to participate in the study through their parents. In Tier I, all consenting children were screened for height, weight, and head circumference, and a large random sample was chosen from all enrolled first-grade children. Every consenting child that was ≤25 centile on head circumference and/or whose height and weight participated in Tiers I and II, along with all randomly-selected children who had been selected from all enrolled children from the same classes and schools, regardless of Tier I pre-screening and size, were included. In Tier II, complete growth, development, and dysmorphology examinations were provided to the above children, both those that were small and randomly-selected ,30. ThenAdditionally, in Tier III, the mothers of all study children were informed of our interest in identifying maternal risk factors for all FASD births, and those who consented to the maternal interview were interviewed about a variety of maternal risk factors. There were many detailed questions about alcohol consumption during the index pregnancy and in their lifetime, and the questions about alcohol access from a variety of sources, including their experience with the Dop system, were embedded in the maternal questionnaire. The questionnaire utilized a time-line-follow-back sequence, along with questions concerning diet, childbearing history and experience, residence, general health, and socioeconomic status. Diagnoses of the children were made according to the first revision of the U.S. Institute of Medicine (IOM) Guidelines . The IOMThe entire IOM continuum of FASD diagnoses is represented by four diagnoses: fetal alcohol syndrome (FAS), partial fetal alcohol syndrome (PFAS), alcohol-related neurodevelopmental deficits (ARND), and alcohol-related birth defects (ARBD) ,31. For Data analysis was performed using SPSS, Version 26 , for allIn In p < 0.001) and that Dop use declined significantly in recent years. There was no significant difference by age category in Dop use percentage during pregnancy, at interview (current use), and by rural residence. Taken together, these data indicate that Dop was rarely reported to have been received during pregnancy at any time over the 15-year period, it was rarely received in recent years, and Dop was still most common in rural areas over the 15-year period. Different drinking patterns were evident in rural vs. urban areas, as presented in In r = 0.117, p < 0.001). The correlation between 5 DDD or more was also significant, but equally weak . Therefore, the associations between Dop and heavy drinking explained only 1.4%–1.5% of the variance in heavy drinking. Additionally, when the average DDD was added to the other two controlled variables above and the correlation of lifetime Dop with an FASD diagnosis was examined, the correlation was even weaker , explaining 0.35% of the variance in FASD diagnosis.In an attempt to place any direct contribution of Dop to maternal drinking and the prevalence of FASD into a quantitative perspective, partial correlations and sequential regression analysis were performed. Partial correlations of lifetime experience with Dop with drinks per drinking day (DDD) were performed while controlling for residency  and maternal age at index pregnancy. The Pearson product-moment correlation between ever receiving Dop in one’s lifetime with an average of 3 DDD or more was statistically significant, but extremely weak  to the diagnosis of FASD in the offspring. A low maternal BMI, rural residence during pregnancy, and higher gravidity at the index pregnancy all also contribute significantly. However, there is no significant, direct association between ever receiving Dop in one’s lifetime and an FASD diagnosis in the offspring .In These community-specific, time series data support the idea that the Dop system of partial payment for labor via the distribution of wine is now extremely rare in the WCP. Even lifetime rates of experience with Dop were found to be very low in the time-period studied. In the more recent time-periods sampled, an even lower percentage of women reported receiving Dop payments in their lifetime, currently, or during pregnancy. In the latest samples collected from the more remote and rural regions of the province, the lifetime prevalence of Dop payments in the two communities was 2.9%–17.5% in rural areas and 0.6%–8.5% in the urban areas. Dop is less commonly reported by those residing in Community A (a developing community closer to Cape Town), than among those living in more remote rural settings in Community B. By the time of the interview, seven years after the birth of the index children, virtually no Dop was being distributed . Moreover, these recent reports of Dop were provided by only a few mothers, all of whom had children diagnosed with an FASD. Therefore, Dop, as a large, common, and viable institution, is essentially dead in the WCP, and is currently reported as a source of alcohol by less than 1% of the mothers of children with an FASD. While none of the mothers of the randomly-selected children who were found to be functioning normally (controls) reported receiving any alcohol via the Dop system, as we have reported before, commercially-produced and purchased alcoholic beverages constitute virtually all of the alcohol consumed by women of a childbearing age in these communities today ,22,23,28While the partial correlation analysis produced a very weak, but statistically significant, association (explaining <2% of the variance) in heavy maternal drinking, the more sophisticated sequential correlation analysis showed no statistically significant, direct relationship between Dop and an FASD diagnosis in the offspring. There is no real direct association between the current Dop distribution and heavy drinking or with the likelihood of an FASD diagnosis in the offspring. It should, furthermore, be noted that the proportion of women reporting drinking during pregnancy in this paper, 41.4%–74.4%, was exponentially higher than the 3.1% of women who had given birth in the past five years reporting that they had consumed alcohol while pregnant . AdditioThe data presented in this paper also provide much insight into the historic pattern of partial payment for labor via the Dop system and how it has been influential in shaping drinking practices of women today. It has, therefore, been influential in shaping normative patterns of alcohol use and the prevalence and pattern of FASD diagnoses found in contemporary WCP communities today. Until very recently, the bulk of the cases of FAS and PFAS (the more severe forms of FASD) have been found in the rural areas where Dop practices were originally implemented, and heavier drinking is still reported by the rural respondents. Therefore, the relationship today is certainly an indirect one transmitted through normative drinking patterns of the Dop heritage to that of today. Other factors, such as the possibility of a cohort effect producing reduced drinking levels in the study areas independently of any effects of the Dop system, are unlikely and not supported by any data. Nevertheless, this cannot be completely excluded. While not much has been written on the effect of the Dop system and its legacy on male health outcomes, besides a paper by London et al. , the depFirst, the pattern of heavy drinking that is present today in the study area, particularly on weekends and in rural areas, appears to be an extension or progression of the old pattern practiced under Dop. Second, 93%–98% of the alcohol consumed by the female respondents in these samples is consumed, on average, on only two days during weekends. Third, DDD remains consistently higher in the rural areas of both regions. DDD averaged 3.9 to 5.5 in Community A, which was more urban, and 5.4 to 5.7 in the more remote and rural Community B. Even though an alcohol allotment was supplied each evening to male workers on some farms under the Dop system, binge drinking was also reported to be a common practice for recreational drinking among men and women under the Dop system on some farms. Such recreational drinking primarily occurred on weekends and holidays. Fourth, few mothers reported drug use until the most recent samples in both communities. There is no appreciable  pattern of increase or decline in drug use reported in Community A over the 15 years studied, and the two samples in Community B were too close together to depict a definitive trend. Given that alcohol is still the drug of choice, the contemporary pattern of drug choice is similar to that of Dop. Fifth, drinking patterns remain more severe in the rural areas, which may be a Dop residual, and the most lifetime experience with Dop is reported by mothers of children with FASD. Sixth, high rates of total FASD have shifted from rural to urban areas in both communities. Although FAS rates have gone down overall, a relative upturn has now been registered in the urban rates in Community A over time. In Community B, the same trend may be emerging. Sixth, lifetime experience with Dop was significantly higher among mothers of children with FASD. Therefore, Dop practices appear to have been influential in directly encouraging heavy drinking that led to FASD in 10%–16% of the mothers in these study communities, especially in the earlier years in Community A, and in 2009 and 2011 in the more isolated Community B. Seventh, and lastly, rates of FAS have declined in the rural areas of Community A and appear to be declining in rural areas of Community B. FASD rates have increased overall in both communities over time, but part of this overall increase is likely due to two factors: increased access to relatively inexpensive, commercially-produced alcohol and the better diagnosis of less severe diagnoses with the FASD continuum . In the most recent samples of both communities, total FASD rates are increasing and higher in the urban areas. Therefore, each of these seven contemporary patterns of alcohol consumption seems to mirror patterns developed in the era of Dop. At least two phenomena may be at work to create the change in the rural and urban distribution of FASD cases over time. Community efforts at FASD prevention were initiated in 1998 in Community A. A National Institute on Alcohol Abuse, and Alcoholism (NIAAA)-funded comprehensive prevention program was also initiated in Community A in 2008 by the NIAAA research and prevention team. In the latter program, much emphasis was placed on awareness training, case management of high-risk women, and targeted education on many of the farms of the rural areas. Prevention efforts may have reduced the heaviest drinking among a significant number of rural women, which in turn may have lowered the severity of FASD characteristics and traits, thereby reducing the rate of FAS, and possibly total FASD among rural dwellers ,23,35. FOne major difference in the drinking style practiced in the Western Cape is a change of beverage. In the days of Dop, the alcoholic beverage most commonly consumed was wine, but in this study period, the beverage of preference was beer. The most frequently purchased beverage was higher alcohol-content beer (approximately 5.5% in the most popular brand) ,22. FurtFirst, the data used in this targeted study were initially collected to determine the prevalence and characteristics of FAS and other FASD in these communities. Therefore, not all Dop questions were asked in the first sample of Community A. However, in all subsequent samples, the full battery of three Dop questions was asked. Second, the study was limited to covering only females of a childbearing age, and the Dop questions were not asked to men, specifically the fathers of the study children. It could be that male workers in the agricultural system were more likely to receive Dop distributions. However, from the information provided by our field workers, we are not aware that this is the case. Third, we know that over the last three or four decades, the majority of farmers and their families are knowledgeable and concerned businessmen and women. Many have increasingly incorporated improvements in permanent residential and working conditions, and the lives of workers have substantially improved on the farms . Where tThe systematic process of Dop is virtually gone or dead, but the legacy of alcohol as the recreational drug of choice remains. Furthermore, the pattern of heavy drinking among men and women is, in many ways, similar to the weekend pattern during the days when Dop was a relatively common institution in WCP. Dop seems to have created a common, culturally-supported pattern of the heavy consumption of commercially purchased alcoholic drinks practiced by most men and many women that has been translated into contemporary practices and, therefore, led to continuing high rates of FASD in these communities.


A short video embed showing the patient riding a bike, running, and squatting has been removed from the article as the video was removed from YouTube.


Today, the adoption of minimal invasive gynecologic procedures is expanding their routine use in clinical practice. Until recently, a diameter of 8 cm was the recommended maximal size for laparoscopic removal of fibroids. However, robot-assisted laparoscopy improved the capacity and the feasibility of the many gynecologic procedures. Here, we report a video of robotic myomectomy of a huge myoma. To demonstrate the feasibility of robotic myomectomy of a huge fibroid, we recorded a robotic myomectomy operation video of a 19 cm diameter (FIGO type 3-4) myoma (Canadian Task Force Classification III) at a university-affiliated private hospital.A 38-year-old, gravida 2  patient with a 19 cm intramural fibroid was admitted to our clinic with a request of endoscopic removal of the fibroid. The patient was given detailed information about risk of the surgery, defining the risk of disseminating malignant cells through the abdominal cavity. It was then decided to perform a myomectomy operation using a robotic platform. The operation was performed using a Da Vinci Xi platform ; the patient card was docked centrally, and three robotic arms and an assistant port with a smoke evacuator  were used.The surgical time (skin to skin) was 205 min, and the docking time was 6 min. A 2.0 barbed suture was used for uterine closure. The estimated blood loss  was 350 cc, and two erythrocyte suspension transfusions were given after the operation. The first gas discharge was 13 hours after the surgery, the length of hospital stay was 2 days. No complications occurred peri-operatively.Huge fibroids can be removed using robot-assisted laparoscopy.Video 1. DOI: 10.4274/jtgga.galenos.2019.2019.0029.video.1