Incurable inflammatory breast cancer (IBC) patients occasionally suffer from general symptoms such as breast pain, bleeding, ulceration, and discharge, and thus require palliative radiotherapy (RT). Hypofractionated RT has many advantages in palliative settings, but very few studies on IBC have been conducted. This study was conducted to evaluate the effects of hypofractionated RT on symptomatic IBC patients.Twenty-two patients with IBC who underwent hypofractionated palliative RT between 2010 and 2016 were retrospectively analyzed. RT was performed at a total dose of 42.5–55 Gy with 2.5–3 Gy per fraction. The treatment effects were evaluated with respect to symptom improvement, tumor response, and treatment-related toxicity.The main symptoms that the patients complained of before RT were pain, bleeding, and discharge. According to the percentage of symptom relief compared with pre-RT symptoms, the number of patients with < 30, 30–70%, and ≥ 70% were 2 (9.1%), 7 (31.8%), and 13 (59.1%), respectively. Eighteen (81.8%) patients showed tumor response. No patient experienced grade 3 or higher acute or chronic toxicity during a median follow-up period of 13 months. In univariate analysis, symptom type was a significant factor for predicting the degree of symptom relief. Meanwhile, RT field and C-reactive protein increase were significant factors for predicting the incidence of radiation-induced skin toxicity.Hypofractionated RT could safely and effectively relieve symptoms among incurable symptomatic IBC patients. Inflammatory breast cancer (IBC) is a rare disease that accounts for 0.5–2% of all invasive breast cancers . The staIBC is known for having more radio-resistance than non-IBC; hence, some studies have suggested more aggressive radiation strategies such as increases in the total radiation dose or changes in the fractionation schedules , 8. In pTherefore, it is necessary to analyze the effects of hypofractionated RT on IBC. Here, we retrospectively analyzed the treatment efficacy and toxicities of incurable and symptomatic IBC patients who underwent hypofractionated RT.Patients who underwent palliative-intent hypofractionated RT for symptomatic IBC between January 2010 and February 2016 at Seoul St. Mary’s Hospital, Seoul, were selected for this analysis. Patients with any of the following conditions were included in this study: 1) presence of clinicopathologically proven IBC based on the 8th edition American Joint Committee on Cancer TNM staging system , 15, 2) This study was a retrospective, with no informed consent from individual patients, but was performed in accordance with relevant guidelines; the study protocol was approved by the intra-institutional ethics committee of the Seoul St. Mary’s Hospital (assigned number: KC15RISI0260).20Gy of < 30%. The heart was limited to V35Gy of < 30% (VxGy: the percentage of the organ volume receiving x Gy or more).All patients were immobilized with vacuum bags in the arm-up position and computed tomography (CT) scans of their breast were taken. These CT images were imported into Pinnacle treatment planning system, Version 9.1 . In RT planning, whole breast and skin were not routinely targeted. The principle of target delineation was as follows. The gross lesion responsible for breast symptoms was defined as the gross tumor volume. This volume sometimes included the adjacent metastatic axillary lymph nodes if it was directly connected to the primary tumor. The clinical target volume was not delineated because treatment was delivered with palliative intent. Subsequently, a volumetric margin of 1.0 cm was applied to create the planning target volume (PTV). If PTV overlapped with the lung or heart, it was manually modified to exclude these organs. Basically, RT plan was designed using 6 MV tangential fields with wedges. Bolus was applied every other day to cover PTV. Two patients for whom the primary mass extended up to the supraclavicular area were also treated with a single anterior oblique photon field. All patients were prescribed a total dose of 42.5–55 Gy with 2.5–3 Gy per fraction, once per day, 5 days a week, for 3–5.5 weeks. In these modest hypofractionated regimens, the total dose was 69.1–89.4 Gy biologically effective dose (BED), which was calculated assuming an alpha/beta ratio of four for breast cancer . The RT P values < 0.05 were considered statistically significant.Before starting RT, all patient symptoms were recorded on a 0–10 numeric rating scale. During the course of RT, patients were evaluated weekly to assess the degree of symptom relief and treatment-related toxicities. After the end of the RT, patients were generally followed-up at 1 week, then every 2 months for the first year, and then every 6 months for the next 3 years. Symptom relief was quantified by the patient as a percentage of symptom relief compared to their baseline. The degree of symptom relief was divided into three categories by modifying Agarwal et al. study  as perceA total of 22 patients were included in this study. Table After approximately 1 month of RT, palliative chemotherapy or hormone therapy was performed in 77.3% (17/22) of patients. The median follow-up duration was 13.9 months , and six patients were alive at the end of the follow-up period. The median OS was 17.3 months, and 1-year and 2-year OS rates were 62.8 and 34.5%, respectively.p = 0.02) was a significant clinical factor for predicting the degree of symptom relief. Patients with a single symptom had significantly better symptom relief than those with overlapping symptoms. In addition, although not statistically significant, symptom relief tended to be better in patients who received a high radiation dose .The main symptoms requiring palliative RT were pain , bleeding , and discharge . Eight patients had two or more combination of symptoms. Stratification for symptom relief  was performed according to the percentage of symptom relief in comparison with the initial symptoms, and the results are listed in Table p = 0.04) and CRP increase during the RT period (p = 0.03) were significant factors for predicting the incidence of radiation-induced skin toxicity.Treatment was well-tolerated. Based on CTCAE criteria, acute complications during and shortly after RT were grade 1 skin dermatitis (faint erythema and dry desquamation) in six patients (27.3%) and grade 2 skin dermatitis (moist desquamation) in 16 patients (72.7%). No patient developed grade 3 or higher skin toxicity and none died from treatment-related toxicity. Chronic complications of radiation, severe chest wall fibrosis, rib fracture, arm edema, and symptomatic pneumonitis were not observed in any patient. These side effects in patients were improved by appropriate supportive care such as frequent dressing, application of topical agents, and collaboration with a dermatologist when necessary. To quantitatively analyze the degree of skin dermatitis, changes in CRP values before, during, and after the RT period were measured. The normal CRP value was 0–5 mg/L. In four patients (18.2%), the CRP level gradually increased during the RT period and fell to the normal range 1 month after RT. The median CRP values before (1 week before RT), during (2 weeks during RT), and after the RT period (1 month after RT) were 6.3, 5.9, and 2.5 mg/L, respectively. Figure We retrospectively analyzed 22 patients who received hypofractionated RT for symptomatic IBC. Most patients showed symptom relief with acceptable skin toxicity. Although OS was much poorer than that of patients who underwent curative-intent standard multimodality treatment, 81.8% of patients showed a tumor response within the follow-up period.It is uncommon to encounter unresectable breast cancer patients in this era when screening tools have been developed and neoadjuvant chemotherapy can be performed. Despite not being tumor resected, RT still plays an important role in women suffering from ulceration, bleeding, or pain in locally advanced and recurrent breast cancer , 21. AltTo date, no study has focused on hypofractionated palliative RT of IBC patients; hence, it is difficult to undertake a direct comparison of the palliation effect. However, indirect comparisons with existing non-IBC studies are possible. Richard et al.  reportedRT-related toxicity was influenced by both fraction size and a wide variety of factors. Bristol et al. reported 10–20% grade 3 or higher treatment-related toxicity in IBC patients receiving curative-intent aggressive multimodality treatment . In our This study has several limitations. The first was the retrospective nature of the study, which may have resulted in heterogeneous patient characteristics particularly among 40.9% of patients who had received previous chemotherapy or hormone therapy, and the second was the relatively small number of cancer patients. However, our study is meaningful in that it focused on patients with IBC as well as on the effects of hypofractionated palliative RT. In future, further studies with more patients will be required to confirm the effect of hypofractionated palliative RT on the quality of life of patients or on complications seen later during the long-term follow-up. In addition, it is possible to conduct studies to examine the increase in local control and disease-free survival by adding aggressive treatment to patients who respond to hypofractionated RT.Our results suggest that hypofractionated RT was relatively effective fo the mono-symptom of breast pain in incurable and symptomatic IBC patients. In addition, this treatment was safe and well tolerated with acceptable toxicity. Further studies to determine an appropriate RT scheme, such as dose fractionation schedule and RT field, are warranted to balance the effects and toxicity.


The mechanism of the mineralization process induced by natural mineralized collagen (MC) has been investigated for decades. The purpose of this study was to investigate the efficacy of self-assembled MC for peri-implant bone defect reconstruction in a mini pig. A standardized peri-implant bone defect model was created using 14 mini pig mandibles. Two materials were evaluated, i.e. a mixture of hydroxyapatite and collagen , and self-assembled MC . Bio-Oss (BO) and untreated  groups were used as controls. After 3- and 6-month healing periods, the mini pigs were sacrificed for histomorphometric and microcomputed tomography analysis. After 3 months of healing, the average alveolar ridge height was 3.27 ± 1.57 mm for group TA, 3.28 ± 2.02 mm for group TB and 3.37 ± 1.09 mm for group BO, while group BC showed the lowest height of 2.68 ± 0.47 mm. After 6 months of healing, the average alveolar ridge height was 2.64 ± 1.13 mm for group TA, 4.31 ± 1.80 mm for group TB and 3.87 ± 1.38 mm for group BO, while group BC showed the lowest height of 2.48 ± 1.80 mm. The experimental groups and control group showed similar bone volume density, bone complexity and histological reaction. The self-assembled MC (Type B) stimulated new bone formation in the reconstruction of deficient alveolar ridges around the dental implant; it also displayed excellent clinical operability compared with bone grafts without collagen. Dental implant restoration is becoming increasingly popular for tooth-loss patients. Sufficient bone volume is needed to guarantee the long-term success of dental implant placement , 2. Howein vivo and the ostointegration is not always ideal [There are four major types of graft materials commonly used for alveolar regeneration application, i.e. autografts, allografts, xenografts and alloplasts. Although an autogenous bone graft obtained from the same individual for ridge repair is considered the gold standard , the souys ideal .et al. [Cui et al.  designedet al. . Additioet al. . HoweverThe Type A (TA) and Type B (TB) materials were prepared and provided by the School of Materials Science and Engineering of Tsinghua University and Beijing Allgens Medical Technology Co., Ltd., respectively. The TA material was a 7/3 mixture of HA and collagen, with a partially MC component. The TB material also had a HA/collagen ratio of 7/3 with MC. The nanosized HA was periodically and orderly arranged between collagen molecules, which self-assembled to form the basic structural units of the MC. Bio-Oss (BO), which is bovine bone calcined at high temperature, was purchased from Geistlich Pharma AG (Switzerland). It was free of all organic components and was used in the control group. The blank control (BC) group contained only bone defects with a collagen membrane , and did not contain any graft-filling material.Fourteen female mini pigs, aged 12 months and with an average body weight of 35 kg, were used in the present study. The study protocol was approved by the Animal Care and Use Committee of Peking University. The animals were anesthetized by injection of pentobarbital sodium (30 mg/kg body weight). After disinfection of the surgical site with chlorohexidine solution (0.2%), local infiltration anesthesia was given. The continuous incision in the gingival crevicular was conducted, the full thickness mucoperiosteal flap was carefully elevated, both side premolars and first molar of lower mandible were sectioned and separated using a high-speed-turbine tooth drill, and carefully extracted from both hemimandibles. The teeth extraction socket was carefully examined to ensure there is no root remnants were left. The mucoperiosteal flap was closely sutured with absorbable suture line.After the 3-month healing period, the tooth extraction sites were reopened. Peeling off the mucoperiosteal flaps revealed the crest and buccal mandible bone. All drilling was done using copious amounts of sterile saline solution. After preparation of the implant cavity for implant insertion, a flat-head fissure bur with diameter 1.5 mm was used to create a standardized acute defects at the buccal bone of the implant cavity under constant irrigation with saline solution. The size of the standardized triangular-shaped defect is 6 mm apicocoronally, 12 mm mesiodistally and 2 mm buccolingually see . Bone-leThe pigs were sacrificed at 3 and 6 months after implant insertion and ridge augmentation. Euthanasia was performed with an overdose of pentobarbital sodium. Subsequently, the implant and supporting tissues were harvested from the jaws and fixed in 10% buffered formalin for 4 weeks.Microcomputed tomography  was used to evaluate the volume of remaining materials after degradation and new bone formation. Images were acquired at an effective pixel size of 8.82 μm, voltage of 80 kV, current of 500 μA and exposure time of 1500 ms for each of 360 rotational steps. The volume of new bone formation and height of the bone augmentation sites were calculated using an Inveon Research Workplace (Siemens).The samples were series dehydrated in 50–99% alcohol solution. Finally, the dehydrated samples were embedded in autopolymerizing methyl methacrylate resin . The resin-embedded specimens were oriented parallel to the longitudinal axis of the implant. One central section was cut in the buccolingual direction (300-μm thick) using a diamond saw , then adhered to the resin slides and ground to approximately 50-μm thickness. The ground slices were stained with methylene blue trihydrate and Van Gieson stain. The histological evaluation by a light microscope mainly focused on new bone formation, inflammatory reaction and especially, the degradation of materials and the response of surrounding tissues to the implant materials.One mini pig was sacrificed due to excessive anesthesia during the implant operation. Although a soft diet was provided to all of the animals, rupture of the surgical wound was inevitable during the early healing phase. Exposure of the cover screw of the implant was observed in 20 of 80 implants; this occurred in all groups and with similar frequency. All other sites healed uneventfully. The movement and daily behavior of the tested animals were not affected. No animals died during the observation period.Previous research reported that guided bone regeneration (GBR) can enable significant bone formation when used for treatment of dehiscence/fenestration defects around implants . A recenet al. analyzed 7473 applications of graft materials in 6182 patients and confirmed that the reconstruction of peri-implant dehiscence defects was associated with the barrier membranes, rather than the graft materials, used [et al. [et al. [A wide variety of grafting materials are available commercially, in various particle sizes and shapes. Clinical efficacy varies by defect geometry, augmentation technique and grafting materials. The results of the present study indicate similar clinical bone augmentation heights regardless of the origin or type of bone-grafting material. Many clinical studies have reported similar results. Troeltzsch ls, used . The res [et al.  and Jaff [et al.  showed t [et al. , 21. In The bone volume fraction is the ratio of the trabecular bone volume (BV) to the total volume (TV) of the area of interest (BV/TV). In addition to bone quantity, the success of an inserted implant strongly depends on the quality of the surrounding bone , 23. BonBone complexity is the ratio of the bone surface area (BS) to the total bone volume (BV) of the area of interest (BS/BV). It is another parameter for evaluating bone quality. The TB material had the highest average value of all of the groups, but there was no statistically significant difference among them see .A peri-implant bone defect model in mini pigs was developed to investigate the efficiency of self-assembled MC on peri-implant bone defect reconstruction in the dental clinic. The results show that self-assembled MC (Type B) stimulated new bone formation in the reconstruction of deficient alveolar ridges around a dental implant. Furthermore, it displayed excellent clinical operability compared with bone grafts without collagen. This work was supported by Beijing Municipal Science & Technology Commission Projects (No. Z181100002018001).Conflict of interest statement. None declared.


Colorectal cancer is one of the most common cancers worldwide. Laparoscopic colorectal surgery (LCRS) is a frequently used modality. A new development in minimally invasive surgery is robot-assisted colorectal surgery (RACRS).Prospectively collected data of 378 consecutive patients who underwent RACRS or LCRS for stage I–III colorectal cancer from Dec 2014 to Oct 2017 were analyzed. Primary outcome was oncological outcome . Secondary outcomes were distant metastases, overall and disease-free survival, operation time, conversion, length of hospital stay, and intra- and post-operative complications.p = 0.60), the average number of harvested lymph nodes was 16 ± 6 versus 18 ± 7 (p = 0.16), and locoregional recurrence rate in 24 months was 3.8% vs 3.8% (p = 0.99), respectively. In rectal cancer, radical margins were achieved in 89.6% in RACRS group versus 94.3% in LCRS group (p = 0.42), the average number of harvested lymph nodes was 16 ± 8 versus 15 ± 4 (p = 0.51), and locoregional recurrence rate in 24 months was 9.5 versus 5.6% (p = 0.42), respectively. Incidence of metastasis, survival rates, operation time, length of hospital stay, and number of severe post-operative complications measured by Clavien–Dindo scores did not differ between RACRS and LCRS groups. Conversion and intra-operative complication rates were significantly lower in the RACRS group as compared to the LCRS group .206 RACRS  and 172 LCRS  procedures were included. Baseline characteristics were similar. Overall median follow-up time was 15 months (0.2–36). Oncological outcome was similar. In colon cancer, radical margins were achieved in 99.3% in RACRS group versus 98.6% in LCRS group (RACRS is safe in the treatment of patients with stage I–III colorectal cancer. Oncological outcome did not differ between RACRS and LCRS groups. RACRS had lower conversion and intra-operative complication rates. Colorectal cancer is one of the most common cancers in the world, the third most common in men, and the second most common in women. Yearly, approximately 700,000 patients with colorectal cancer die worldwide . IntroduBoth open and laparoscopic surgeries have been proven to be safe and laparoscopic resections are associated with several short-term advantages . LocoregA new development in minimally invasive surgery is robot-assisted colorectal surgery (RACRS). Potential benefits of RACRS include improved visualization due to a three-dimensional and magnified image with stable camera platform, advanced dexterity of instruments, and the possibility of the surgeon solely controlling the camera and assist arm, allowing maximal control. These benefits may improve clinical and oncological outcomes in colorectal surgery. Current available literature presumes RARCS to be safe with comparable short-term results as compared to laparoscopic colorectal surgery (LCRS) –12. HoweExpectations for the future are that RACRS will overcome the possible shortcomings of conventional techniques, improving oncological outcomes and reducing complication rates. Therefore, the aim of this study is to enhance knowledge of the oncological and surgical safety of RACRS as a treatment for colorectal cancer.A single-center, longitudinal, prospective cohort study was performed. All procedures have been performed in the Canisius Wilhelmina Hospital (CWH), Nijmegen, the Netherlands. CWH is a large-volume teaching hospital for laparoscopic and robot-assisted surgery. All laparoscopic procedures were performed by dedicated laparoscopic surgeons, and surgeons performing RACRS were certificated for the daVinci robot system . The study was started with the acquisition of the daVinci robot system by the CWH and contains an implementation phase. Peri-operative, pathological, and clinical follow-up data were collected.The study protocol was approved by the local board of ethics of the Canisius Wilhelmina Hospital (CWH).Data on patient, tumor, operative characteristics , neoadjuvant treatment, post-operative complications (short and long term), pathology results , length of hospital stay, readmissions, post-operative mortality (30 days), locoregional recurrence, distant metastases, overall survival (OS), and disease-free survival (DFS) were recorded and analyzed.Resection margins and number of retrieved lymph nodes were evaluated by examining pathologist reports, consisting of the microscopically assessment of pathology specimens. Circumferential resection margin (CRM) was defined positive if malignant cells were found at microscopy at 1 mm or less from the CRM. Proximal and distal resection margins were defined positive if malignant cells of the outermost part of the tumor were found at microscopy at the cutting edge of the tissue.Locoregional recurrence of disease and distant metastases was diagnosed by pathological confirmation after resection or biopsy, or by radiological detection of lesions that increased in size over time. Locoregional recurrence of disease was defined as reappearance of cancer at the primary cancer site or in lymph nodes. Distant metastases were defined as malignant cells that have spread to distant organs, such as liver, lung, or peritoneum.Complications were classified using the Clavien–Dindo classification . Severe Laparotomy for any other reason than specimen extraction from the abdomen was considered as conversion. Operation time was defined as the total time from patient in to patient out of the operating room. Incision time was defined as total time from incision to skin closure.Consecutive patients that underwent RACRS or LCRS for primary resectable clinical stage I–III (T1-3N0-2M0) colorectal cancer between December 2014 and October 2017 were selected for a retrospective analysis of a prospectively collected database. Exclusion criteria were patients who were not suitable for minimal invasive approach , emergency surgery, or operation due to locoregional recurrence. Whether a patient could be distributed to RACRS or LCRS was solely dependent on the operational schedule and the availability of the daVinci Xi robot.Patients with a minimum follow-up of 6 months in the CWH were analyzed for oncologic outcome parameters, including locoregional recurrence rate, distant metastases, OS, and DFS. Patients with a follow-up of less than 6 months, and patients that choose to participate in follow-up elsewhere were excluded for this analysis. Figure All patients were treated in consistence with the evidence-based Dutch national guidelines 2014 [The procedures were totally laparoscopic or totally robotic. Types of surgery performed for colon cancer were left hemicolectomy, right hemicolectomy, transverse colon resection, sigmoid resection, or low anterior resections. Conventional medial-to-lateral and lateral-to-medial approaches were used in accordance with the preference of individual surgeons. Surgery performed for rectal cancer was low anterior resection or abdominoperineal resection with dissection of the rectum according to total or partial mesorectal excision principles. The need for abdominoperineal resection was determined by the tumor characteristics not being separable from external sphincter or pelvic floor structures. Procedures that needed opening of the peritoneal reflection and partial mesorectal excision for radical removal of the tumor were classified as low anterior resections, independent of the tumor localization .Port placement in LCRS group was according to general standardized laparoscopic principles. In the RACRS group, a 12-mm camera port was placed at the umbilicus. In addition, three 8-mm robotic instrument ports were placed along an imaginary linear line, starting from the subxiphoidal space to the left or right iliac region. The distance between each port was at least 8 cm to reduce external collisions. One additional laparoscopic trocar was placed in the contralateral flank to assist if necessary. The anastomoses were performed tension-free and either intracorporeally (circular or linear stapled) or extracorporeally (handsewn or linear stapled) dependent on the surgeons’ choice. In robot-assisted procedures, all anastomoses were performed intracorporeally. Indication for protective loop ileostomy was low colorectal anastomosis; however, the final choice was left to the decision of the surgeon.The daVinci Xi Surgical System  was used for all robot-assisted procedures.Post-operative care was performed consistent with the early recovery after surgery (ERAS) protocol .Follow-up moments were 6, 12, 18, 24, and 36 months after surgery with blood tests and imaging according to the evidence-based Dutch national guidelines 2014 [X2 test was used to compare categorical variables and the independent-samples T test was used to compare continuous variables. For comparison of locoregional recurrence of disease, distant metastases, DFS, and OS, the Kaplan–Meier method was used and its results were compared with the log-rank test. All data were analyzed using IBM SPSS statistics 24. P values of less than 0.05 were considered significant.The In total, 378 surgical procedures for stage I–III colorectal cancer were included. Two-hundred and six RACRS (129 (63%) colon and 77 (37%) rectal) and 172 LCRS (138 (80%) colon and 34 (20%) rectal) procedures were analyzed.Baseline patient characteristics did not differ between RACRS and LCRS groups Table , and thip < 0.001), and significantly more right-sided  and left-sided  colon resections performed in LCRS group. For rectal cancer, the distribution was equal  of the rectal tumors was located too close to the anal verge for low anterior resection  and needed abdominoperineal resection.The mean operation and incision time was similar in RACRS group and LCRS group for both colon and rectal procedures, and no significant differences were identified.p = 0.008). There was no significant difference in the colon resections: 4 conversions versus 10 conversions in RACRS and LCRS groups (p = 0.129), respectively. For rectal cancer, 2 conversions in RACRS group and 6 conversions in the LCRS group were performed (p = 0.005).Six (2.9%) RACRS procedures were converted to open surgery compared to 16 (9.3%) LCRS procedures (p = 0.003). Intra-operative complications all occurred during colon resections in the RARCS group and in 10 colon (p = 0.062) and 3 rectal (p = 0.008) resections in the LCRS group. The intra-operative data are shown in Table Intra-operative complications occurred in 3 out of 206 (1.5%) of RACRS procedures and in 13 out of 172 (7.6%) of LCRS procedures  months. Figure p = 0.592). The mean harvested lymph nodes was 16.4 ± 5.9 versus 17.6 ± 7.3 (p = 0.155), respectively.Radical resection margins were achieved in 133 (99.3%) tumors in RACRS group compared to 139 (98.6%) tumors in LCRS group  in LCRS group. The mean harvested lymph nodes was 16.0 ± 8.0 versus 15.3 ± 3.8 (p = 0.506), respectively.Radical resection margins were achieved in 69 (89.6%) tumors in RACRS group compared to 33 (94.3%) tumors  with conventional LCRS. Several retrospective studies with mainly small sample sizes have evaluated the short-term outcomes of robot-assisted surgery for colon cancer , 16–21 aRadical resection margins and number of lymph nodes harvest are important factors in the assessment of quality of surgery. Excision of malignancies with appropriate tumor-free margins is considered curative. In rectal cancer, circumferential resection margin (CRM) involvement is a predictor of poor prognosis, increasing the risk of locoregional recurrence of disease by 3.5 times . In the p = 0.419), respectively. Similar to the present study, two comparative studies suggest equal locoregional recurrence rate for rectal cancer, of which one prospective analysis [In terms of recurrence of disease, regular follow-up showed no significant difference between RACRS and LCRS groups in the first 24 months after surgery. Local recurrence rate in colon cancer was 3.8% for both RACRS and LCRS groups and in rectal cancer 9.5% versus 5.6%  and intra-operative complications (p = 0.003) was identified in the LCRS group. In current study, reasons for conversion were mainly anatomic and tumor-specific reasons (5/6 in RACRS group and 9/16 in LCRS group). In LCRS group, 7 patients were converted due to insufficient view or complication, compared to only 1 in RACRS group. Additionally, in LCRS group, injury of nearby organs  occurred 9 times, but none of these events occurred in RACRS group. Similarity in patient characteristics and clinical and pathological cancer stages argue against a reflection of selection bias. Improved visualization and advanced control in robot-assisted surgery could have prevented occurrence of conversions and complications in RACRS group. Furthermore, improved intra-operative outcomes in robot-assisted surgery are also earlier reported. A prospective analysis performed by Baik et al. [Surgical safety of robot-assisted procedures is as important as the oncological safety to approve its use in colorectal cancer. Hospitalization time and complication rates were similar between RACRS and LCRS groups. A higher rate of conversion (k et al.  of 56 rok et al.  of 765 rThe present study had some limitations. First, this study was not double-blinded or randomized. It was a retrospective analysis of a prospectively collected database and therefore subject to selection bias, associated with this type of study design. This resulted in a distribution of colon and rectal cancer procedures that was not similar between RACRS and LCRS group. However, patients were all operated in the same period with similar peri-operative treatment protocol. Clinical characteristics of patients, indications of operation, and pathological cancer stages were similar between RACRS and LCRS group. This similarity persists for colon and rectal cancer procedures separately. Supplementary analyses were performed to identify confounding. For intra-operative and pathological outcome, additionally, robot-assisted procedures were compared to laparoscopic procedures for colon and rectal resections separately. On the other hand, a smaller number of surgeons were experienced with RACRS procedures compared to LCRS procedures. Therefore, there was a wider range of surgeons performing LCRS, possibly making the procedure more variable with different preferences in surgical approaches. By obligation of nationally approved laparoscopic surgical skills training for all surgeons and an additional training module for the surgeons performing RACRS, an adequate, minimal level of surgical skills was established. Small differences, however, cannot be ruled out. Furthermore, the current study was started in the implementation phase of daVinci Xi in the CWH. Internal analysis showed a learning curve of 32 interventions for operation time for an individual surgeon. No learning curve effects were found regarding clinical outcome parameters. Therefore, operation time in RACRS group might be shorter than observed in the full study period, but no other biases are expected.The median follow-up time of the present study was relatively short to make definitive comparisons of oncological follow-up data. However, oncological follow-up of robot-assisted surgery has rarely been described in the literature. Kang et al.  describeIn conclusion, RACRS is safe and feasible in the treatment of primary resectable stage I–III colorectal cancer. Oncological outcome did not differ with similar radical resection margins, lymph nodes harvested, and locoregional recurrence during 24 months after surgery compared to LCRS. Benefits of RACRS have been noticed for operative outcomes, i.e., less conversion rate and intra-operative complications. Furthermore, similar operative times have been observed for RACRS and LCRS.Future clinical trials with daVinci Xi should be realized to confirm current evidence in favor of RACRS and the suggestions of shorter operation times. Furthermore, extended research is needed to determine whether equal oncological outcomes will last in long-term follow-up.


Patients completed a STwalk, and 4 conditions with 1-back (low load) and 2-back (high load): STlow, SThigh, DTlow, and DThigh. Overground walking was performed at a comfortable pace and -N-back conditions were carried out verbally. Both gait  and cognitive (rate of correct answers) performances were recorded. Changes in PFC oxyhemoglobin (ΔO2Hb) and deoxyhemoglobin (ΔHHb) were measured by functional near infrared spectroscopy (fNIRS). Results showed an increase of ΔO2Hb while walking, which was not augmented by cognitive loads in DT. Walking speed was reduced by low and high cognitive loads in DT compared to STwalk (P < 0.05), but was not different between DTlow and DThigh. Cognitive performances were negatively impacted by both walking (P < 0.05) and cognitive load . These data highlight a “ceiling” effect in ΔO2Hb levels while walking, leaving no available resources for simultaneous cognitive tasks, during the early recovery period following stroke. In these patients, cognitive, but not motor, performances declined with a higher cognitive load.Stroke patients often exhibit difficulties performing a cognitive task while walking, defined as a dual task (DT). Their prefrontal cortex (PFC) activity is higher in DT than in single task (ST). The effects of an increasing load on PFC activity during DT in subacute stroke patients remains unexplored. Our objective was to assess the effects of N-back tasks (low/high load) on cerebral activity, gait parameters, and cognitive performances. Eleven subacute stroke patients (days post-stroke 45.8 ± 31.6) participated in this pilot study (71.4 ± 10 years, BMI 26.7 ± 4.8 kg.m Stroke patients with brain lesions may exhibit impaired cognitive functions, altered walking capacity, or both Grotta, . These d2Hb) levels in stroke patients, have reported greater changes in the prefrontal cortex (PFC) during DT than in single task (ST) , although a few studies proposed that the imaging of ankle dorsi-flexion, a component movement of gait, may provide a useful marker for gait recovery  participated in this pilot study, in the center of Physical Medicine and Rehabilitation . Inclusion criteria for post-stroke patients included: acute (<2 weeks after stroke) or early subacute stroke (between 2 weeks and 3 months) HAS, , first sThe study was approved by national ethic committee  and patients gave their informed consent.low and SThigh), walking single task  and DT including simultaneous cognitive and walking conditions . Cognitive tasks for ST and DT followed a modified N-back from Fraser et al. , stride variability , and stride asymmetry , the percentage of correct answers was compiled for each condition, as missing or incorrect answers were accounted for as errors. The DT costs on cognition and gait were calculated by:Patients performed walking conditions  on an 8-m GAITRite walkway , which provided spatio-temporal gait parameters, such as speed (Cognitive costs:low/high is the cognitive performance (percentage of correct answers); andwhere Score DTGait costs:low to DThigh are, respectively given by the equations:The cognitive and gait costs from DT2Hb and HHb  in the test interval  were then normalized by subtracting to them the mean value of the last 10 s of baseline, immediately before the beginning of the task, i.e., seated for STlow and SThigh, and standing for STwalk, DTlow, and DThigh. From these data were extracted the hemoglobin difference (ΔHbDiff = ΔO2Hb − ΔHHb) and the laterality index (LI) defined as the ratio:Cerebral oxygenation was measured using a fNIRS system . Two optodes were placed on symmetrical prefrontal sites Fp1 and Fp2 according to the EEG 10/20 system. Acquisition was made through the Oxysoft software (version 3.0.97.1). Differential Pathlength Factor was set on 5 as its calculation formula does not apply to patients' age 50 years and older  and cognitive load  on cerebral activity  and gait parameters . A Spearman correlation test was then conducted to establish potential correlations between PFC activity and gait/cognitive performances. The statistical significance was set at P < 0.05.A Shapiro-Wilk test confirmed the non-normality of data. Friedman and Wilcoxon tests were then conducted to compare and assess the respective effects of walking  or between cognitive conditions. No effect of cognitive load was observed for other oxygenation parameters, such as ΔHHb.There was a main effect of walking with an increase of ΔO2Hb in the respective affected and unaffected hemispheres (P < 0.01) whereas LI was not modified .Finally, there were no significant correlations between PFC activation  and gait/cognitive performances  was superior to cgDTlow (respectively −19 ± 18% and −15 ± 19%) but walking did not significantly influence the cognitive cost .The gait cost of cognitive load in “high” condition  was −58 ± 31% (P < 0.01) but cgDTlow → high was negligible.The cognitive cost of heavier cognitive load during DT , as observed in other studies in stroke patients .The very limited number of patients included in this study was due to the difficulty of performing both walking and cognitive tasks in subacute phase. In parallel, it is important to note that fNIRS studies during walking show technical and methodological risks which can modify results in study findings if they are not controlled  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki.AP and JD: study concept and design. BT and EH: acquisition of data. EH, OD, and AP: analysis and interpretation of data. EH, AP, and JD: drafting of the manuscript. SF, MC, and JS: critical revision of the manuscript for important intellectual content.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Colletotrichum sublineolum. An in vitro batch culture study model with different carbon sources, glucose, arabinose and rhamnose, were used to support fungal growth over a period of twelve days. Metabolites representing the intracellular and extracellular (secreted) metabolomes were extracted with methanol and subjected to LC-MS analyses. Chemometric modelling revealed a metabolic variation trajectory, comprising three distinct stages that metabolically describe the adaptation of the fungus to diminishing nutrients. Selected marker gene expression indicated stage one (0–3 d.p.i) as corresponding to the early logarithmic phase. Stage two can be interpreted as an intermediate transitionary stage with stage three corresponding to the stationary phase (9–12 d.p.i). Stage one was characterised by up-regulation of endo-metabolites such as ferricrocin, fatty acids and flavone-conjugates, while stage three was characterised by the secretion of phytotoxins, including colletotrichin and colletotric acid. Ultimately, results from our in vitro model reveal previously unknown insights into the dynamic aspects of metabolome reprogramming in the growth phases of Colletotrichum spp as determined by nutrients obtainable from plant cell walls.Metabolomics has emerged as a powerful approach to comprehensively interrogate cellular biochemistry. As such, we applied an untargeted liquid chromatography-mass spectrometry metabolomic strategy to elucidate metabolome changes in the anthracnose-causing hemibiotrophic sorghum pathogen,  Colletotrichum is one of the most widespread and economically detrimental genera of plant pathogenic fungi, and represents a serious threat to global food security and ecosystem health2. Members of this genus are etiological agents of anthracnose leaf spot and other diseases including blights and post-harvest rots on a vast range of agronomic and horticultural crops4. The severe damage caused by Colletotrichum spp leads to major losses in economically important crops, extending particularly to vital staple foods in tropical and sub-tropical regions5.Colletotrichum pathology characteristically follows a multistage hemibiotrophic infection strategy involving the formation of a series of specialised cell types6. Following adhesion of spores, melanin-pigmented appressoria are formed and mediate initial host penetration using mechanical force and enzymatic degradation. Furthermore, Colletotrichum fungi differentiate specialised infection vesicles, which then begin the growth of primary hyphae that develop inside living host cells surrounded by an intact host plasma membrane7. This symptomless stage of infection, i.e. biotrophy, is short lived and the fungus switches to a necrotrophic stage, forming differentiated and morphologically distinct secondary hyphae that kill and destroy host tissue for nutrient acquisition. The lifestyle transition involves dynamic multi-layered reprogramming, including the expression of a vast array of genes encoding lytic enzymes, transporters and secondary metabolism-related enzymes as well as toxin biosynthesis that accompanies rapid growth, systemic colonisation and breakdown of host cells9.Colletotrichum fungi, and have provided substantial knowledge of the underlying infection strategies10. Furthermore, considering high diversity and tractability to in vitro culture, Colletotrichum spp provide excellent models for studying the molecular mechanisms and biochemistry of infection structure differentiation and fungal-plant interactions12. However, there is still much to uncover about the chemical signalling and regulatory mechanisms underlying the transition from biotrophy to necrotrophy of Colletotrichum pathogens that may allow the development of novel approaches to control fungal diseases in crop plants.Extensive studies have been carried out using cytological, physiological, genomic and transcriptomic approaches to elucidate the molecular details that govern the lifestyles of hemibiotrophic in planta are technically challenging and represents a specific challenge to metabolomic approaches due to the close association between plant and pathogen, coupled with a general commonality of metabolites13. Here, we opted for an in vitro culture system, coupled to a liquid chromatography-mass spectrometry (LC-MS)-based untargeted metabolomic approach to gain information about the underlying biochemistry associated with the different growth stages of C. sublineolum. This was achieved by profiling the methanol soluble secondary - and some primary metabolites present in the endo- and exometabolomes that characterise the different stages of growth in batch culture. The uncovered metabolic signatures, accompanied with expression levels of selected marker genes, identified specific biochemical processes involved in the growth stages of the fungus, generating a holistic molecular description of the trophic transition phenomenology.Studies of fungal growth 14, can provide informative insights about the biochemical underpinnings governing fungal adaptation to the host environment15. Glucose, polymers of glucose and related derivatives are main constituents of plant cell walls and are also present in the apoplast and intracellular compartments17, while arabinose and rhamnose are neutral sugars in the pectic polysaccharide fraction19. Previous studies have indicated that arabinose and rhamnose stimulate endopolygalacturonase gene expression in C. lindemuthianum, important for establishing the fungal infection by targeting the pectin polymers in the cell wall and middle lamella15. Here, following a reductionist approach, different carbon sources namely glucose, arabinose and rhamnose15 were added to Murashige and Skoog (MS) culture medium to provide a controlled in vitro nutrient environment for C. sublineolum growth. This basal salt medium was selected as it does not contain any organic compounds as nutrients that could interfere with subsequent downstream metabolomics analyses. C. sublineolum could grow in the culture media containing these different carbon sources (with a slow rate in rhamnose-containing media)15 with a noticeable carbon assimilation . The PCA modelling indicated stability, reliability and reproducibility of the analyses – as confirmed by the quality control (QC) samples clustering closely in the center of the plot –, and natural separation between control and inoculated samples  methanol-soluble metabolite extracts were analysed on an LC-MS analytical platform, allowing the simultaneous detection of multiple analytes  with high sensitivity. Chromatographically, the resultant differential profiles  revealed by PC analyses (i.e time-related sample groupings in scores space) 23 was utilized to descriptively explain these time-related metabolic changes in C. sublineolum growth revealed by PCA, treating each carbon source  as an individual batch, comprising a series of times . BP/M provided an efficient means of infographically visualising the biochemical response to the type of carbon source in terms of both inter-group variation and net variation in intra/extracellular metabolite profiles. BP/M allowed for the evolution of the metabolic changes to be statistically characterised and described in time trajectories and provided thus a template for defining the sequence of time-dependent metabolic profiles. Significant time-related changes could subsequently be identified, as well as metabolic features  related to these changes.As mentioned, once host cells have been penetrated, ce) Fig.  and 9. Hm/z, Rt variables: spectral descriptors) and the metabolite collection time (the maturity variable) was decomposed by subjecting it to two subsequent levels of multivariate analysis: batch evolution (or lower level) and batch level (or upper level) modelling24. The former is based on PLS regression against metabolite collection time, thus providing a descriptive characterisation of the metabolic variation trajectory in terms of PLS components. PLS takes advantage of the correlation that already exists between the LC-MS variables  and time; generating scores plots that explain the greatest variance in the data with respect to time.The three-way data matrix built up by the batches (carbon source conditions), the pre-processed LC-MS variables (Y (time), according to cross-validation, and indicated a significantly substantial relationship of X-variables with time  were mapped to generate a time-course trajectory plot describing the different phases of the dominant metabolic perturbations with time , which progressively explained less of the covariance in the data and orthogonal to each other, were also plotted over time to establish more subtle, time-related metabolic variations  until the last time point, and no deviating behaviour (outside the ±3 SD control limits) was observed.The computed batch evolution (lower level PLS) models were statistically significant and reliable, with a good predictability in ime Fig. . Since time Fig. . Lower se , wt[2]/t[1] vectors) , transition (3–6 d.p.i.) and stationary (6–12 d.p.i.). These mathematically elucidated developmental time-periods of fungal growth correlate to previous studies, which reported that the biotrophic phase of Colletotrichum pathogens is short-lived, lasting about 72 h post-infection/inoculation, followed by the switch to necrotrophic lifestyle12. However, very little substantial knowledge exists with regards to the underlying biochemical and molecular mechanisms and differences in growth stages do not necessarily correspond to biotrophy or necrotrophy. Hence, it could be possible that there is actually a transition period (as this in vitro study suggests: 3–6 d.p.i.) that can inform on the in planta situation where the fungus gradually responds to environmental cues and adapts to a complete necrotrophic phase.Furthermore, the  average PLS trajectory plotted in two dimensions (rs) Fig.  clearly actin as a reference gene. Quantitative measurements were normalised to give the relative gene expression wherein error bars represent the standard error of mean (SEM)  Fig. .Figure 4Colletotrichum graminicola6, namely sodium/hydrogen exchanger family protein (NaH), GMC oxidoreductase (Oxi) and zinc carboxypeptidase (Zinc), showed a significant up-regulation at 3 d.p.i., independently of carbon source, where after the expression levels decreased from 6 d.p.i., with a significant decrease in expression levels at 12 d.p.i. , pectate lyase (Pec) and putative peptidase family M28 (Ppep) were significantly expressed at 9–12 d.p.i. , the expression of these genes could be seen also at 6 d.p.i.  during the in vitro growth, development and differentiation of C. sublineolum in batch culture.Despite some carbon source-related differential gene expression levels observed, the results indicate that the 0–3 d.p.i. time interval is associated with group 1 gene expression, whereas group 2 gene expression is linked to the time period of 9–12 d.p.i. Fig. . Althoug.i. Fig. . Except .i. Fig. , the timvs. – 9–12 d.p.i.), were of diverse metabolic origins and biochemical functions during fungal growth  between the measured significant metabolites. The constructed correlation-based networks .Lumichrome , a breakdown product of riboflavinAspergillus spp, Fusarium spp, and Laetisaria arvalis28. Moreover, nonanoic acid (pelargonic acid) is reported to weaken the waxy cuticle of plants, causing cell disruption and cell leakage . These properties might contribute to the establishment of a C. sublineolum infection and penetration associated with its biotrophic phase.Furthermore, the stage 1 metabolite profile was characterised by hydroxylated free fatty acids, - amide and - dioic acid conjugates and derivatives Table . Scienti32. The transition element, iron, is essential in key metabolic processes31 and plays a vital role in plant-microbe interactions34. A study on the related hemibiotrophic maize pathogen C. graminicola, revealed that siderophores are required for the establishment of vegetative growth of the fungus, and are also involved in modulating the plant immune system35.Important signatory biomarkers related to the early growth phase were the siderophores, namely ferricrocin and coprogen . This finding is consistent with previous studies that indicated that the biotrophic phase of Colletotrichum spp is marked by activation of a genomic program involved in secondary metabolism, directed also to toxin production20.Notably, the polyketide, 5-methoxysterigmatocystin, Table , Fig. 5 C. sublineolum  and late growth stages (exemplified by up-regulation and secretion of toxins). This observation correlates to a highly complex and coordinated genetic and transcriptional program, as well as capacity deployed by Colletotrichum in its infection strategy as previously reported; involving activation of genes related to degradative enzymes, expression of secondary metabolism gene clusters, metabolite diversity via transcriptional regulation8. Thus, this predictive description proposes that the Colletotrichum lifestyle is metabolically more complex  than currently understood, spanning various metabolic pathways.It can be postulated that with the significant depletion of carbon sources , obtainable in planta as cell wall- and pectin-derived nutrients, provided insights into the metabolic changes in C. sublineolum, suggesting specific metabolic features during the growth course of the fungus.For successful infection of host plants, hemi-biotrophic fungal pathogens like the C. sublineolum during the transition from stage 1 to stage 3 growth. By applying a dynamic multivariate method, the time-course phases were elucidated over a period of 12 days. Stage 1 was seen to be short lived (0–3 d.p.i.), followed by a transition interval, stage 2 (3–6 d.p.i.), leading to stage 3 (9–12 d.p.i.). Stage 1 was accompanied with the expression of genes reported to be associated with biotrophy while stage 3 was associated with expression of genes associated with necrotrophy in C. graminicola6.Our results chemometrically describe that metabolic reprogramming occurs in in planta situation.The metabolic profiles of these growth stages of the fungus were characterised by reprogramming of primary and secondary metabolism, with production of phytotoxins in stage 3. The metabolic states, as uncovered by both the endo- and exo-metabolome profiles, point to complex metabolomic changes that might be of functional significance to the interaction between the pathogen and host in an vs. stage 3 would imply that endogenous signals related to nutrient depletion are necessary and sufficient to trigger the changes in gene expression associated with necrotrophy. However, care should be taken in extrapolating the in vitro growth results to biotrophic – and necrotrophic stages in infected plant tissues. Also, this model, does not address the possibility that other signals, originating from fungal structures in the infected plant or from the plant itself, might play contributing roles in such a lifestyle switch. However, it does lay a solid foundation for further targeted metabolomic studies related to specific stages of fungal growth and development, in vitro or in planta.The marker gene expression during stage 1 C. sublineolum in vitro growth stages thus pointed to specific biochemical processes and deciphered regulatory mechanisms, providing a holistic description of growth and adaptation to diminishing nutrient conditions. Such knowledge contributes to enhance insight into the infection strategies of the fungal pathogen and the dynamic intricacies of the molecular interactions between Colletotrichum spp and plant targets. As such, the present work provides a necessary step for in planta studies, uncovering metabolic central hubs and key drivers in molecular events governing the C. sublineolum biology. Finally, the metabolomic information presented in this work will be a valuable resource for relating data from other omics layers towards systems biology description of the phenomenology of plant-fungal pathogen interactions. Colletotrichum spp represent economically significant pathogens where a holistic and detailed understanding of pathogen biology could substantially inform strategies for targeted disease control and management.Unravelling metabolite profile patterns underlying the C. sublineolum (PPRI 7183) was grown and maintained on potato dextrose agar (PDA). The working sub-cultures were maintained on half-strength PDA solid media in Petri dishes and incubated under a 12 h–fluorescent light cycle at 25 °C. To simulate in planta nutrient sources, three types of Murashige and Skoog -based liquid media  containing 10 g L−1 of different carbon sources, namely glucose, arabinose and rhamnose, were prepared under sterile conditions42.A pathogenic isolate of C. sublineolum grown on solid PDA media (14 day-old cultures). The flasks were incubated at 25 °C on a rotary shaker at 130 rpm under a 12 h–fluorescent light cycle. Consumption of glucose during the growth stages of the fungus was quantitatively assessed using a glucose measuring device  on supernatants separated from the mycelial bio-mass harvested at 3, 6, 9 and 12 days post-inoculation (d.p.i.)  were each inoculated with three mycelial plugs (2 mm diameter) from The experiment was conducted in three biological replicates. For extracellular samples, non-inoculated liquid media was also incubated and harvested under the same conditions, to account for any change that might have occurred in the media over time and served as negative controls.The organic solvents used, methanol and acetonitrile, were UHPLC-MS grade quality , and water was purified by a Milli-Q Gradient A10 system . Leucine enkephalin and formic acid were purchased from Sigma Aldrich/Merck .44.Each flask was harvested by filtering the contents through a double layer of Miracloth under vacuum to separate the fungal mycelia and the media. The mycelia samples were weighed and the wet-weight recorded. The extracellular metabolites (the exo-metabolome: metabolites secreted into the media) were extracted from 25 mL freeze-dried media filtrate with 50% methanol and filtered through a 0.22 µm nylon filter into glass chromatography vials fitted with 500 µL inserts. The intracellular metabolites (endo-metabolome) were extracted using 100% methanol added directly to the weighed-out fungal material in a ratio of 1:10 (w/v), and immediately homogenised using an Ultra Turrax homogeniser followed by sonication using a probe sonicator , set at 55% power for 20 sec and repeated twice, which served to rapidly quench enzymatic activitiesg for 15 min at 4 °C. The supernatants were placed in 50 mL round-bottom flasks and evaporated to 1 mL at 50 °C using rotary evaporation under vacuum, and dried to completeness with a speed vacuum centrifuge at 45 °C. The dried residues were re-constituted in 300 µL 50% methanol and filtered through a 0.22 µm nylon filter as described. The filtered aqueous-methanol extracts prepared from the endo- and exo-metabolomes were stored at −20 °C until analysed.The homogenates were then centrifuged at 5000 × 3, v/v) (eluent A) and formic acid: acetonitrile  (eluent B) were pumped at 0.4 mL min−1 into the UHPLC system. The applied gradient started at 5% B and increased linearly to 95% B over 22 min. The conditions were kept constant for 3 min to wash the column and returned to initial conditions at 27 min. The analytical column was allowed to equilibrate for 3 min. Each sample was analysed in triplicate to account for any analytical variability and the run time for each analysis was 30 min. The LC system was coupled to a SYNAPT G1 high definition (HD) quadrupole time-of-flight (Q-TOF) mass spectrometer , with an electrospray ionisation (ESI) source. The TOF analyser was used in V-optics, and the data were acquired in centroid mode.Chromatographic separation of metabolites was performed on an Acquity UHPLC system  using a conditioned autosampler at 4 °C. Two µL of methanol-extracted samples were separated on an analytical C18 column HSS T3 , thermostatted at 60 °C. Degassed solutions of formic acid: ultra-pure water  simultaneously by collision energy ramping from 10 to 40 eV. The software used to control the hyphenated system and perform all data manipulation was MassLynx 4.1 .Both positive and negative ionisation modes were employed with the scan range of 100–1000 Da, a scan time of 0.2 s and an inter-scan delay of 0.02 s. A lock spray source was used allowing online mass correction to obtain high mass accuracy of analytes . Leucine enkephalin, [M + H]46. Sample acquisition was randomised and the QC sample (6 injections) analysed every 10 injections to monitor and correct changes in the instrument response. Furthermore, 6 QC runs were performed at the beginning and end of the batch to ensure system equilibration. Such sample randomisation provides stochastic stratification in sample acquisition so as to minimise measurement bias. In the principal components analysis (PCA) space, the QC samples were clustered closely to each other  pooled samples were used to condition the LC-MS analytical system so as to assess the reliability and reproducibility of the analysis, and for non-linear signal correctionher Fig. , thus coTM 4.1 software  for both the centroid ESI positive and negative raw data. The MarkerLynxTM application manager of the MassLynx software was used for data pre-processing (matrix creation), producing a matrix of retention time (Rt)-m/z variable pairs, with m/z peak intensity for each sample. The parameters of the MarkerLynx application were set to analyse the 1–25 min Rt range of the mass chromatogram, mass range 100–1000 Da, and alignment of peaks across samples within the range of ±0.05 Da and ±0.20 min mass and Rt windows, respectively; and mass tolerance of 0.01 Da. The MarkerLynx application uses the patented ApexPeakTrack algorithm to perform accurate peak detection and alignment. Following the peak detection, the associated ions are analysed  and captured for all samples. In this study, sample normalisation was done by using total ion intensities of each defined peak. Prior to calculating intensities, MarkerLynx performs a patented modified Savitzky-Golay smoothing and integration.Visualisation and data processing were performed using MassLynx XSThe data matrices thus generated were exported into SIMCA  software, version 14  for chemometric analyses, including multivariate and univariate statistical modelling, and an unsupervised method, namely PCA, was employed. The data pre-treatment methods used included Pareto-scaling, and as described in the results section, the computed models were rigorously validated.23, allowing a multivariate statistical description of time-related events. In a BP/M approach, PLS regression analysis against time (i.e. the time period over which the changes are recorded) allows for the evolution of the metabolic changes to be statistically characterized and described in time trajectories. Thus, significant time-related changes can subsequently be identified, as well as metabolic features  related to/that explain these changes24.A partial least squares (PLS)-based approach, namely batch processing/modelling (BP/M), was used to optimally assess and describe the time-related metabolic trajectories observed from PC-analyses48. Structural similarities were determined based on similarities between PubChem Substructure Fingerprints (ftp://ftp.ncbi.nlm.nih.gov/pubchem/specifications/pubchem_fingerprints.txt). The R package Chemminer generated molecular fingerprints using the PubChem Power User Gateway (PUG). Molecular fingerprints in the form of ordered lists of binary bits defining presence or absence of physical properties  were used to calculate structural similarities; pairwise similarities were calculated based on Tanimoto similarity between two bit vectors resulting in scores between 0 and 1, where a score of 0 or 1 defines no or complete overlap in structural properties between the two molecules. A Tanimoto coefficient cut-off of 0.5 was used for network analyses.Biochemical networks were generated using CytoscapeTM-based data processing were exported to the Taverna workbench (www.taverna.org.uk) for PUTMEDID_LCMS Metabolite ID workflows49. The Taverna workflows consist of correlation analysis, metabolic feature - and metabolite annotation and allow for integrated, automated and high-throughput annotation and putative metabolite identification from LC-ESI-MS metabolomic data. The Taverna Metabolite ID procedure involves: (i) Pearson-based correlation analysis (List_CorrData), (ii) metabolic feature annotation (annotate_Massmatch) – allowing for grouping together ion peaks with similar features such as Rt, and annotating features with the type of m/z ion  believed to originate from the same compound. The elemental composition/molecular formula (MF) of each m/z ion was then automatically calculated; and (iii) metabolite annotation (matchMF-MF) of the calculated MF (from the output file from workflow 2) was automatically compared and matched to the MF from a pre-defined reference file of metabolites.Data matrices from MarkerLynxwww.dnp.chemnetbase.com), Chemspider (www.chemspider.com), Knapsack database (http://kanaya.naist.jp/KNApSAcK/) and KEGG (www.genome.jp/kegg/); (ii) structural confirmation through careful inspection of fragmentation patterns by examining the MS1 and MSE spectra of the selected metabolite candidate. Metabolites were annotated to level 2 as classified by the Metabolomics Standard Initiative (MSI)50.For confidence in metabolite annotation, the following steps were performed: (i) the calculated MF of a selected metabolite candidate was manually searched against databases and bioinformatics tools, such as the Dictionary of Natural Products (DNP)  and the integrity verified by denaturing agarose gel electrophoresis.Total RNA was extracted from 100 mg mycelia of all time point samples using the Trizol-reagent  and then subjected to DNase treatment using DNase I , per the manufacturers’ instructions. Concentrations were determined using a NanoDrop® ND-1000C. graminicola6, six C. sublineolum genes were selected for gene expression analysis in C. sublineolum grown in a culture containing either arabinose, glucose or rhamnose as carbon source. The selected marker-genes for the biotrophic phase were zinc carboxypeptidase (JMSE01000325.1), putative sodium/hydrogen exchanger family protein (JMSE01001189.1) and glucose-methanol-choline (GMC) oxidoreductase (JMSE01001299.1); while the genes for the necrotrophic phase included fungal cellulose binding domain-containing protein (JMSE01001209.1), pectate lyase (JMSE01001554.1) and putative peptidase family M28 (JMSE01001300.1). The primer pairs (Supplementary Table www.ncbi.nlm.nih.gov/genbank). Prior to quantification of the expression levels, the DNase-treated RNA was reverse-transcribed to cDNA using a RevertAid™ Premium First Strand cDNA synthesis kit .Based on the putative function during biotrophic and necrotrophic development of ase JMSE0000325.1,qPCR was performed to analyse the expression of each gene on a Rotor gene-3000A instrument  using the SensiFAST SYBR No-ROX Kit  according to the manufacturer’s instructions. Ten μL SensiFAST SYBR, 1 μL  each forward and reverse primers, and 6 μL DNase-free water was added to 2 μL cDNA for amplification in a total volume of 20 μL. The cycling conditions were as follows: initial denaturation for 2 min at 95 °C followed by amplification and quantification cycle repeated 40 times each consisting of 5 s denaturing at 95 °C, 10 s annealing at primer specific temperatures, and 20 s extension at 72 °C. Two biological replicates were used with two technical replicates of each.51 was used to quantify the expression levels of the selected genes and the data normalised using actin (ref.6 and Supplementary Fig. p < 0.05 were considered significant.The relative standard curve methodTime-resolved decoding of metabolic signatures of <Emphasis Type="Italic">in vitro</Emphasis> growth of the hemibiotrophic pathogen <Emphasis Type="Italic">Colletotrichum sublineolum</Emphasis>


Renal cell carcinomas (CCR) account for 90% of renal tumors. Presence of latent distant metastasis is characteristic of RCC and may manifest more than a decade after nephrectomy. Gallbladder (GB) is a rare site of metastasis, with few reports in the literature. A case of metastasis from RCC to GB nine years after initial diagnosis is reported herein.Male patient, 74 years, nine years post right radical nephrectomy for grade 2 clear-cell adenocarcinoma, T3BN0M0. During onset, Magnetic Resonance Imaging (MRI) evidenced T2-weighted hypointense and T1-weighted hyperintense lesion, with early and persistente contrast enhancement and exophytic bulging of the underlying outer vesicular margin. T1-weighted hypointense and T2-weighted slightly hyperintense nodular formation was also evidenced in the body portion of the pancreas, with 1.5 × 1.2 cm. The patient was subjected to videolaparoscopic cholecystectomy associated to endoscopic ultrasound (EUS) intraoperatively for investigation of the pancreatic nodule. The anatomopathological examination of the gallbladder was compatible with infiltrating metastasis from clear-cell carcinoma of primary renal site. A solid, hypoechoic, oval nodule with 14 mm was found at EUS, which cytology was suggestive of clear-cell Carcinoma. Because this is an indolent disease with oligometastasis, local ablative treatment with fractionated stereotactic radiation therapy with a dose of 40 Gy was selected. The patient is found with stable disease one year after radiation therapy.Gallbladder is an unusual site of RCC metastasis. In patients with history of this disease, all vesicular lesions should be given attention, even where the primary tumor has been treated many years before. Its prevalence has increased in the recent years and the presence of latent distant metastasis is characteristic of RCC and may manifest more than a decade after nephrectomy. Clear Cell (CC) RCC is the most common type of renal cancer, accounting for 75% of all primary kidney tumours [Gallbladder (GB) is a rare site of metastasis, with few robust reports in the literature containing clear descriptions of imaging, surgical and anatomopathological parts that add information for its recognition ,3. The c2Male patient, 74 years old, nine years post right videolaparoscopic radical nephrectomy for grade 2 clear-cell adenocarcinoma, T3BN0M0 (not subjected to systemic chemotherapy), during annual onset on private practice setting, it was found a gallbladder polyp with 0.7 × 0.7 cm on computed tomography (CT). He was completely asymptomatic. After one year, in 2017, CT was repeated with evidence of polyp growth to 1.7 × 1.3 cm. Investigation was complemented with Magnetic Resonance Imaging (MRI), which evidenced T2-weighted hypointense and T1-weighted hyperintense lesion, with early and persistent contrast enhancement and exophytic bulging of the underlying outer vesicular margin, showing irregular contours A and B. One month later, the patient was subjected to videolaparoscopic cholecystectomy associated to endoscopic ultrasound (EUS) intraoperatively for investigation of the pancreatic nodule.The anatomopathological examination of the surgical specimen - gallbladder  was compIMAGE 1A: Expansive formation on the right lateral body wall of the gallbladder, with 1.7 × 1.3 cm, showing pronounced early and persistent contrast enhancement and promoting exophytic bulging of the underlying outer vesicular margin, which shows irregular contours (Red circle). 1B: T2-weighted hypointense expansive formation in the right lateral body wall of the gallbladder (Yellow circle) and T2-weighted slightly hyperintense nodular formation in the body portion of the pancreas (White circle).IMAGE 2: T1-weighted hypointense nodular formation in the body portion of the pancreas with 1.5 × 1.2 cm (circle).IMAGE 3: Metastatic renal cell carcinoma as a well-circumscribed polypoid mass in the gallbladder body (circle).IMAGE 4: Gallbladder with areas of mucosal erosion (arrow) and metastatic neoplastic process constituted of wide and clear cytoplasm cells, permeating the wall of the organ (circle).IMAGE 5: Immunohistochemistry A: Neoplastic cells evidencing CD10 immunolabeling positivity. B:Neoplastic cells evidencing “Renal Cell Carcinoma” (RCC) immunolabeling positivity.3RCC is one the most lethal tumors of the urological system. Its five-year survival rate in all stages is approximately 69% . This isGB tumors are often diagnosed as polypoid masses and have a wide range of differential diagnoses. The most common are primary vesicular adenomas and adenocarcinomas. Metastases in this organ are rare events in the clinical practice, with primary stomach tumors, melanomas and RCC being the main origins .CT is the most common imaging method in oncology for planning the therapeutic strategy; however, even with this tool, differentiation between primary vesicular tumors and metastatic lesions is still difficult ,9, with There are about 50 RCC metastasis to the gallbladder reported in the literature, from 1963 to the present day, but a significant part of them was diagnosed at autopsy ,10,11 thUnlike primary gallbladder carcinoma, RCC metastases are predominantly found in male patients, with low incidence of gallstones association . Most caRCC metastasis to the gallbladder occurs by the systemic dissemination of the disease, not by contiguity, since its appearance is late (average of four years post-nephrectomy ), the meA review of RCC metastasis conducted in 2012 evidenced the presence of RCC metastasis to the gallbladder and pancreas in 21% of the patients analyzed, even surpassing lung metastasis . Lung isA systematic review in 2015 defined a follow-up algorithm for gallbladder polyps. Based on the risk of malignancy found, polyps greater than 10 mm should be submitted to a surgical procedure, polyps smaller than 4 mm can be observed, with follow up every 2 or 3 years, and polyps between 4 and 10 mm should have their probability of malignancy calculated based on the following criteria: single polyp, sessile polyp and patient age. Polyps with a probability of malignancy greater than 18% should also be resected [4Gallbladder is an unusual site of RCC metastasis; therefore, its diagnosis deserves no active investigation in patients with history of the disease. However, all vesicular lesions in this population should be given attention and surgical treatment should be considered, regardless of the size of the lesion, even when the primary tumor has been treated many years before.No conflict of interest relevant to this article was reported.At our own expenses.Ethics approval: Santa Casa de São Paulo Ethics and Research Comittee in 05/11/2018.: 01571818.5.0000.5479.Reference numberWritten informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.Mauricio A. Ribeiro: Conceptualization and Methodology; Bruno H. Lucia and Fabio Kater: Investigation, Data collection; Fabiana Bueno: Resources; Maria Carolina Galli Mortati: Writing – Original Draft; Caroline Petersen da C. Ferreira: Writing – Review & Editing and Visualization; Luiz A. Szutan: Supervision and Project Administration.Case reports that are not first-in-man study already approved in Ethics Committee.Caroline Petersen da Costa Ferreira.Not commissioned, externally peer-reviewed.


Eribulin improves survival in pre-treated HER2-negative advanced breast cancer (ABC). However, limited data exist on co-morbidities and central nervous system (CNS) efficacy. The purpose of this study was to review eribulin’s efficacy and safety in everyday clinical practice with special focus on age, body mass index (BMI) and central nervous system (CNS) activity.An observational study was conducted in a series of HER2-negative ABC patients treated from January’14-December’17 outside a clinical trial. Objective Response Rate (ORR), Progression Free Survival (PFS), Overall Survival (OS), and association of clinical and pathological variables with outcome were evaluated.Ninety-five women were treated with at least one cycle of eribulin. Median age was 57 (33–83), and 18% were obese. Median number of prior chemotherapies for ABC was 3 (2–5) and 76% of patients had visceral metastases, including 21% with CNS involvement. Most tumors were estrogen receptor-positive (79%). ORR and stable disease (SD) at 6 months were 26.2 and 37.5%, respectively. Remarkably, relevant CNS efficacy was observed with eribulin: 20% of patients obtained partial response and 25% SD. Treatment was generally well tolerated and manageable, with 29% grade 3 and 10.9% grade 4 toxicities. Median PFS and OS were 4.1 months (CI95% 3.2–4.9) and 11.1 months (CI95% 9.5–14.7), respectively. Triple-negative disease, > 2organs involved and being younger than 70 years old were independent prognosis factors for worse OS in multivariate analysis. Most patients (75%) progressed in pre-existing metastases sites.In everyday clinical practice, eribulin’s efficacy seems similar to pivotal trials. CNS-efficacy was observed. TNBC, > 2 organs involved and being younger than 70 years old were independent prognosis factors for worse OS. Remarkably, less incidence of grade 4-toxicity compared to previous studies was found. Eribulin mesylate is a synthetic analogue of the marine halicondrin B, a microtubule-targeting agent approved for the treatment of metastatic breast cancer. Some preclinical data showed that eribulin can reverse the epithelial–mesenchymal transition (EMT), reducing cancer cell migration and invasion , 2. AddiTwo phase III randomised studies  and a pooled analysis of these two studies showed that eribulin improved overall survival (OS) in patients with pretreated advanced breast cancer (ABC) –8. The bEribulin was approved in Spain in December 2013 for patients with HER2-negative recurrent or metastasic breast cancer previously treated with taxanes, anthracyclines and capecitabine unless patients were not suitable for those treatments –19. The An observational study was conducted at ICO-Hospitalet to review patients with HER2-negative ABC consecutively treated with single-agent eribulin from January 2014 to December 2017 in an off-trial setting. All patients included may have received at least one cycle of eribulin 1.23 mg/m2 day 1 and 8 every 21 days. The treatment was continued until disease progression, unacceptable toxicity, or patient withdrawal. Age at diagnosis, body mass index (BMI), relevant comorbidities, Eastern Cooperative Oncology Group (ECOG) Performance Status, tumor subtype, visceral involvement, pre and post-eribulin chemotherapy regimens, response rate, clinical benefit, CNS efficacy, toxicities and pattern of progression were obtained from medical records. Toxicity was reported following the Common Terminology Criteria for Adverse Events (CTCAE 3.0).Regarding comorbidity, we did not use the Charlson Comorbidity Index due to the strong influence of the presence of metastases (M1) on this scale, but presence of relevant comorbidity used in that scale was registered and graded, and we considered it relevant if patients had one or more comorbidities with grade 2 or higher. In this study the comorbidity that was considered and graded was: diabetes mellitus, hypertension, dyslipidemia, myocardial disease such as myocardial infarction and congestive heart failure, chronic pulmonary disease, liver and renal disease.The characteristics of patients were described and the Kaplan–Meier method was used to calculate the PFS and OS as the time-to-event for each endpoint from the start date of treatment with eribulin to the occurrence date of the event, progression or death, respectively. An evaluation of the response was performed by the response evaluation criteria in solid tumors (RECIST 1.1.). Objective response rate (ORR) included complete response (CR) and partial response (PR) at the first evaluation . Clinical benefit rate included CR, PR or stable disease (SD) of at least 6 months’ duration.p-value of < 0.05 was considered significant. In the multivariate analysis, we included the variables that achieved a p-value < 0.1 in the univariate analysis. All analyses were carried out using the SPSS 22.0 statistical software .Statistical differences were estimated using the log-rank test. The univariate and multivariate analyses were conducted using the Cox proportional hazard regression model for PFS and OS to assess the prognostic value of patients, tumors and treatment characteristics. A two-tailed n = 20), seven (33%) had been previously treated with whole brain irradiation. In patients pretreated with taxanes, 46% progressed during the first 3 months and they were considered taxane-refractory, based on previous studies [Ninety-five women with HER2-negative ABC were treated with at least one cycle of eribulin during this period. Baseline patient and tumor characteristics are listed in Table  studies .Table 1Median number of cycles of eribulin received was 7 (1–55). Dose interruptions, delays and reductions were 14, 26 and 32%, respectively. ORR was 26.2%  and 37.5% of patients had SD, with a clinical benefit observed in 63.7% of patients. Regarding CNS efficacy, 4 patients (20%) presented a PR, 5 patients (25%) achieved SD and 2 patients were not evaluated. Remarkably, out of the nine patients with CNS metastases who progressed during treatment with eribulin, six (66%) had meningeal carcinomatosis. In the subgroup of patients without previously known CNS disease, only two patients progressed to meningeal carcinomatosis during treatment with eribulin. Disease control seemed independent to previous CNS local treatment. From the seven patients who had been previously treated with whole brain irradiation, two achieved PR, three SD and two progressed during treatment with eribulin.p = 0.299), with 25% vs 31.3% achieving PR and 37% vs 20% achieving SD, respectively.A different pattern of ORR was observed in patients based on age (< 70 years vs ≥70 years). Clinical benefit was 62% in the younger group of patients and 51.3% in the older patients, non-statistically significant (p = 0.01); in patients with TNBC compared to those with HR-positive tumors ; in patients with visceral involvement ; and in patients < 70 years of age . Median OS was 11.1 months (95% CI 9.5–14.67) and, similar to what was observed with PFS, OS was significantly shorter in patients with TNBC compared to those with HR-positive tumors  ; in patients with more than 2 organs involved  ; in patients with visceral involvement  ; and in patients < 70 years of age  . Median OS was worse for taxane-refractory patients  and better for patients who experienced grade 3 or 4 toxicities , but these differences were not statistically significant. No differences in PFS or OS were observed based on comorbidity or weight.At a median follow up of 13 months, 16 patients (17%) were still alive. Median PFS was 4.1 months (95% CI 3.2–4.9) and was significantly shorter in patients with more than 2 organs involved , > 2 organs involved  and < 70 years of age  were independent prognostic factors for worse OS  progressed in pre-existing sites of metastases and only 19 (20%) patients experienced disease progression in new locations. Four patients out of those 19 (21%) developed meningeal carcinomatosis. The remaining 5% of patients had clinical progression without radiological evidence of disease progression. The main cause of treatment release was progression (87%) followed by toxicity (13%). Median number of lines of chemotherapy received after eribulin was 1 (0–5).Most patients  –8. PatieIn previous reports, patient age had not been described as an independent factor for PFS or OS. A pooled analysis of 827 patients treated in either the EMBRACE trial or the preceding phase II studies reported outcomes according to age. A total of 10% of patients who were included in these studies were aged ≥70 years. No significant differences were observed in ORR, PFS, or OS by age. Toxicity was similar across all age groups, although the incidence of grade 3 and 4 fatigue and peripheral neuropathy was highest in patients aged ≥70 years , 21. EldThe role of BMI and comorbidities in the outcomes of patients treated with eribulin is still a matter of debate. In some historical series, patients who are overweight and obese have shown worse outcomes compared to the normal weight group , 23, 24.In our study, 21% of patients had CNS involvement, which is often an exclusion criterion in most clinical trials, unless patients are previously treated with local therapy. Of those patients, 25% achieved SD and 20% achieved PR, which supports the efficacy of eribulin in patients with CNS involvement. The efficacy in patients with CNS disease was not related to previous treatment with whole brain radiotherapy. The rationale for potential positive effect of eribulin on brain metastases is not clear. Preclinical evidence suggests that the agent does not cross the blood–brain barrier (BBB) to a significant extent , 26. RadThe pattern of disease progression in patients treated with eribulin is distinctive. Most patients in our series progressed in pre-existing M1 sites (75%). This fact may reflect the inhibitory effect of eribulin in the EMT, reducing cancer cell migration and invasiveness; however, we did not find any information about the pattern of progression in patients who received other oncospecific treatments . It has Regarding toxicity, most patients in our series demonstrated acceptable tolerability of the eribulin treatment. The most frequent grade 3/4 toxicities were asthenia, neurotoxicity and neutropenia and were manageable. Remarkably, the proportion of grade 3 neutropenia was lower in our study than in pivotal trials (9.8% vs 23.4%), as well as grade 4 toxicities with 10.2% in our study  versus 27.7% in the pooled analysis [This study has several strengths and limitations. Our study reflects the everyday clinical practice use of eribulin and analyzes multiple variables such as BMI, age > 70 years and activity in patients with CNS metastases that have not been previously addressed and studied together in patients outside of a clinical trial. The major limitations of this study are the limited number of patients included and its retrospective nature without having a dedicated pre-planned design of patient management and data collection, which can lead to potential biases. It is worth noting that, during the study period, several clinical trials evaluating new targeted drugs in combination with eribulin were ongoing and those patients were not included in this study.The efficacy of eribulin in terms of OS and PFS observed in our study is similar to that reported in pivotal clinical trials. However, clinical benefit and ORR were higher, and significantly longer survival was seen in patients with HR-positive tumors, low burden disease and older than 70 years of age. Remarkably, less toxicity was found compared to previous studies and relevant CNS efficacy was observed. Despite the potential biases of this study, its results might be useful in clinical practice due to the limited existing data of eribulin’s efficacy and safety focused on age, BMI and CSN.


Staphylococcus aureus has served as a model organism for the study of heme synthesis and heme-dependent physiology and, like many species of the phyla Firmicutes and Actinobacteria, generates heme through a coproporphyrin intermediate. A critical step in terminal heme synthesis is the production of coproporphyrin by the CgoX enzyme, which was presumed to be oxygen dependent. However, S. aureus also requires heme during anaerobic growth; therefore, the synthesis of coproporphyrin by an oxygen-independent mechanism is required. Here, we identify CgoX as the enzyme performing the oxygen-dependent and -independent synthesis of coproporphyrin from coproporphyrinogen, resolving a key outstanding question in the coproporphyrin-dependent heme synthesis pathway.Heme is a critical molecule required for aerobic and anaerobic respiration by organisms across kingdoms. The human pathogen  Staphylococcus aureus is supported by many heme-dependent proteins, including key enzymes of cellular respiration. Therefore, synthesis of heme is a critical component of staphylococcal physiology. S. aureus generates heme via the coproporphyrin-dependent pathway, conserved across members of the Firmicutes and Actinobacteria. In this work, we genetically investigate the oxidation of coproporphyrinogen to coproporphyrin in this heme synthesis pathway. The coproporphyrinogen III oxidase CgoX has previously been identified as the oxygen-dependent enzyme responsible for this conversion under aerobic conditions. However, because S. aureus uses heme during anaerobic nitrate respiration, we hypothesized that coproporphyrin production is able to proceed in the absence of oxygen. Therefore, we tested the contribution to anaerobic heme synthesis of CgoX and two other proteins previously identified as potential oxygen-independent coproporphyrinogen dehydrogenases, NWMN_1486 and NWMN_1636. We have found that CgoX alone is responsible for aerobic and anaerobic coproporphyrin synthesis from coproporphyrinogen and is required for aerobic and anaerobic heme-dependent growth. This work provides an explanation for how S. aureus heme synthesis proceeds under both aerobic and anaerobic conditions.The virulence of the human pathogen IMPORTANCE Heme is a critical molecule required for aerobic and anaerobic respiration by organisms across kingdoms. The human pathogen Staphylococcus aureus has served as a model organism for the study of heme synthesis and heme-dependent physiology and, like many species of the phyla Firmicutes and Actinobacteria, generates heme through a coproporphyrin intermediate. A critical step in terminal heme synthesis is the production of coproporphyrin by the CgoX enzyme, which was presumed to be oxygen dependent. However, S. aureus also requires heme during anaerobic growth; therefore, the synthesis of coproporphyrin by an oxygen-independent mechanism is required. Here, we identify CgoX as the enzyme performing the oxygen-dependent and -independent synthesis of coproporphyrin from coproporphyrinogen, resolving a key outstanding question in the coproporphyrin-dependent heme synthesis pathway. Staphylococcus aureus is an important human pathogen responsible for a wide variety of diseases in numerous host niches  that subunit , 5) that subunit , 7) thatS. aureus nitrite reductase  which proceeds to uroporphyrinogen. Uroporphyrinogen can be diverted to form similar cofactors, including siroheme for use in the eductase . Bacterieductase . In onlyrmediate , 12. Thermediate –15. HoweS. aureus and other organisms that synthesize heme via coproporphyrin, UroD produces coproporphyrinogen , supplemented with Casamino Acids and glycerol as the primary carbon source, to quantitatively assess aerobic and anaerobic heme-dependent growth. As glycerol is nonfermentable, growth in this medium relies largely on oxygen-dependent (aerobic) or nitrate-dependent (anaerobic) respiration. S. aureus wild type (WT) grew robustly in this medium in the presence of oxygen, but the heme-deficient ΔcgoX and ΔuroD mutants were unable to grow  or broth (TSB) supplemented with 10 μg/ml chloramphenicol when necessary. When used, hemin chloride (referred to as heme) was used at concentrations noted in the text or on figures. Heme was prepared fresh at 10 mM in 0.1 M NaOH; for experiments in which heme was used, an equal volume of 0.1 M NaOH was used for all conditions. Escherichia coli strains were grown on lysogeny broth (LB) or LB agar (LBA), supplemented with 50 μg/ml carbenicillin when necessary. For growth in aerobic liquid medium, an Innova44 incubator with shaking at 180 rpm was used. For standard cultures of 2 to 3 ml, 15-ml round-bottomed polypropylene tubes with aeration lids were used, at a 45° angle in the aerobic incubator or upright and without shaking in the anaerobic incubator. Unless noted otherwise, all chemicals are from Sigma. All molecular biology reagents were from New England Biolabs and used according to the manufacturer’s instructions, unless otherwise noted. Phusion 2X high-fidelity master mix was used for all PCRs for cloning.For bacterial strains, plasmids, and primers, see the supplemental material  Gene deletions. In-frame, unmarked deletions were created by allelic exchange as described in Bae and Schneewind  and JC421/JC422 (downstream flanking). For cgoX, flanking regions were amplified using primers JC631/JC632 (upstream flanking) and JC633/JC508 (downstream flanking). Deletions of uroD and cgoX were confirmed using primers JC427/JC428. For NWMN_1636, flanking regions were amplified using JC621/JC622 (upstream flanking) and JC623/JC624 (downstream flanking). The deletion was confirmed with PCR using primers JC627/JC628. For NWMN_1486, flanking regions were amplified using JC617/JC618 (upstream flanking) and JC619/JC620 (downstream flanking). The deletion was confirmed with PCR using primers JC625/JC626. To create ΔcgoX ΔNWMN_1636, pKOR1-cgoX was transduced as described previously  Genetic complementation. To complement the ΔNWMN_1636 mutant phenotypes, NWMN_1636 was cloned from S. aureus Newman genomic DNA using primers JC644/JC645 with homology to pOS1 Plgt digested with NdeI and BamHI (NEB) and ligated using NEB HiFi assembly mix. To complement the ΔcgoX mutant phenotypes, cgoX was cloned from S. aureus Newman genomic DNA using primers JC515/JC516 to incorporate a C-terminal FLAG tag with homology to pOS1 Plgt digested with XhoI and BamHI (NEB) and ligated using NEB HiFi assembly mix. The cgoX genes of other species were cloned in the same manner, except that nested PCR was used to accommodate the length of the 3′ primer incorporating the FLAG tag sequence, as noted below. Genomic DNA was used as the template for Firmicutes species, while pET15b-cgoX (S. coelicolor) (provided by the Dailey lab) was used as the template for S. coelicolorcgoX. For S. carnosus, primers JC735/JC736 were used to amplify cgoX from the genome and became the template for JC735/JC737 to amplify the final product; for S. lugdunensis, primers JC738/7JC39 were used to amplify cgoX from the genome and became the template for JC738/JC740 to amplify the final product; for L. monocytogenes, primers JC744/JC745 were used to amplify cgoX from the genome and became the template for JC744/JC746 to amplify the final product; for B. anthracis, primers JC747/JC748 were used to amplify cgoX from the genome and became the template for JC747/JC749 to amplify the final product. S. coelicolorcgoX did not require a nested PCR and was amplified with primers JC791/JC792. This plasmid was sequenced to confirm its accuracy. The complementation plasmids were confirmed by restriction digest after isolation from DH5α following transformation, transformed into S. aureus RN4220 by electroporation, and transduced as described previously (eviously  using ba(iv) Growth curves. Growth curves were performed using overnight cultures of biological triplicates or quadruplicates depending on the experiment. After overnight growth, 1 μl of overnight culture was added to 199 μl of RPMI medium  supplemented with 1% Casamino Acids, 0.04% glycerol, and 40 mM nitrate  in a 96-well round-bottomed plate (Costar), and growth was monitored in a BioTek plate reader with shaking at 37°C. Data were graphed using GraphPad Prism. Three independent experiments were performed , the mean of the biological triplicates or quadruplicates was calculated, and the three means were graphed with the standard errors of the means shown for each figure panel, except for panels in which no error bars are displayed for ease of viewing. The same data, with errors indicated, are presented as supplemental figures. For aerobic growth curves, strains were streaked to TSA and grown aerobically for 24 h at 37°C. Single colonies were used  to inoculate overnight cultures in 3 ml of TSB in aeration tubes and grown for 15 h with shaking at 180 rpm in an Innova44 incubator at 37°C. For anaerobic experiments, a Coy anaerobic chamber was used, filled with a mix of 90% nitrogen, 5% carbon dioxide, and 5% hydrogen gases, and hydrogen levels were monitored to ensure a minimum of 2% hydrogen concentration. Palladium catalysts (Coy) were used to remove any residual oxygen by reaction with hydrogen. A Coy static incubator was maintained at 37°C. Solutions and plasticware were allowed to equilibrate for >24 h inside the glove box before use. For anaerobic samples, strains were streaked to TSA and grown aerobically for 24 h at 37°C. TSA plates were moved into the anaerobic chamber, and single colonies were used  to inoculate overnight cultures in 2 ml of anaerobic TSB in aeration tubes and grown for 16 h anaerobically without shaking at 37°.