Emerging nitrogen-fixing cyanobacteria for sustainable cotton cultivation.

Amid growing environmental concerns and the imperative for sustainable agricultural practices, this study examines the potential of nitrogen-fixing cyanobacteria as biofertilizers, particularly in cotton cultivation. The reliance on synthetic nitrogen fertilizers (SNFs), prevalent in modern agriculture, poses significant environmental challenges, including greenhouse gas emissions and water system contamination. This research aims to shift this paradigm by exploring the capacity of cyanobacteria as a natural and sustainable alternative. Utilizing advanced metabarcoding methods to analyze the 16S rRNA gene, we conducted a comprehensive assessment of soil bacterial communities within cotton fields. This study focused on evaluating the diversity, structure, taxonomic composition, and potential functional characteristics of these communities. Emphasis was placed on the isolation of native N2-fixing cyanobacteria strains rom cotton soils, and their subsequent effects on cotton growth. Results from our study demonstrate significant plant growth-promoting (PGP) activities, measured as N2 fixation, production of Phytohormones, Fe solubilization and biofertilization potential of five isolated cyanobacterial strains, underscoring their efficacy in cotton. These findings suggest a viable pathway for replacing chemical-synthetic nitrogen fertilizers with natural, organic alternatives. The reintegration of these beneficial species into agricultural ecosystems can enhance crop growth while fostering a balanced microbial environment, thus contributing to the broader goals of global sustainable agriculture.

Vaginal Approach to Cesarean Section Scar Pregnancies: A Standardized Ten-step Surgical Procedure.

OBJECTIVE: To show a 10-step standardized vaginal surgical technique to treat first-trimester cesarean section scar pregnancies. DESIGN: A video article with a stepwise demonstration captured in an operation room of a tertiary medical center of a low-income country (Bolivia). SETTING: Cesarean section scar pregnancies have a wide variety of management options. We present a vaginal surgical approach that has been developed by our group with no observed complications in 6 consecutive cases at the time of this publication. INTERVENTIONS: A 10-step technique: (1) patient positioning, (2) cervical exposure and traction, (3) cervical infiltration with vasoconstrictor solution, (4) anterior mucosal incision and dissection of vesicovaginal space, (5) identification of vascular pedicle and ligation, (6) cold-knife isthmocele incision and ectopic pregnancy evacuation, (7) uterine cavity curettage, (8) hysterorrhaphy with interrupted suture, (9) cystoscopy, and (10) vaginal mucosa closure with running suture. We have used this technique in 6 consecutive patients. Operating time ranged between 20 and 25 minutes. All patients were discharged in postoperative day 1. CONCLUSION: We prefer surgical options for these patients because it provides both a complete removal of the ectopic pregnancy and repair of the isthmocele. A vaginal approach to such cases has proven to be a safe, simple, and fully reproductible technique. We find it applicable for any patient with this pathology but especially suitable for low-resource or somehow financially challenged hospitals.

Phylogenetic and functional analysis of cyanobacterial Cytochrome c6-like proteins.

All known photosynthetic cyanobacteria carry a cytochrome c 6 protein that acts transferring electrons from cytochrome b 6 f complex to photosystem I, in photosynthesis, or cytochrome c oxidase, in respiration. In most of the cyanobacteria, at least one homologue to cytochrome c 6 is found, the so-called cytochrome c 6B or cytochrome c 6C. However, the function of these cytochrome c 6-like proteins is still unknown. Recently, it has been proposed a common origin of these proteins as well as the reclassification of the cytochrome c 6C group as c 6B, renaming the new joint group as cytochrome c 6BC. Another homologue to cytochrome c 6 has not been classified yet, the formerly called cytochrome c 6-3, which is present in the heterocyst-forming filamentous cyanobacteria Nostoc sp. PCC 7119. In this work, we propose the inclusion of this group as an independent group in the genealogy of cytochrome c 6-like proteins with significant differences from cytochrome c 6 and cytochrome c 6BC, with the proposed name cytochrome c 6D. To support this proposal, new data about phylogeny, genome localisation and functional properties of cytochrome c 6-like proteins is provided. Also, we have analysed the interaction of cytochrome c 6-like proteins with cytochrome f by isothermal titration calorimetry and by molecular docking, concluding that c 6-like proteins could interact with cytochrome b 6 f complex in a similar fashion as cytochrome c 6. Finally, we have analysed the reactivity of cytochrome c 6-like proteins with membranes enriched in terminal oxidases of cyanobacteria by oxygen uptake experiments, concluding that cytochrome c 6D is able to react with the specific copper-oxidase of the heterocysts, the cytochrome c oxidase 2.

Symbiosis between cyanobacteria and plants: from molecular studies to agronomic applications.

Nitrogen-fixing cyanobacteria from the order Nostocales are able to establish symbiotic relationships with diverse plant species. They are promiscuous symbionts, as the same strain of cyanobacterium is able to form symbiotic biological nitrogen-fixing relationships with different plants species. This review will focus on the different types of cyanobacterial-plant associations, both endophytic and epiphytic, and provide insights from a structural viewpoint, as well as our current understanding of the mechanisms involved in the symbiotic crosstalk. In all these symbioses, the benefit for the plant is clear; it obtains from the cyanobacterium fixed nitrogen and other bioactive compounds, such as phytohormones, polysaccharides, siderophores, or vitamins, leading to enhanced plant growth and productivity. Additionally, there is increasing use of different cyanobacterial species as bio-inoculants for biological nitrogen fixation to improve soil fertility and crop production, thus providing an eco-friendly, alternative, and sustainable approach to reduce the over-reliance on synthetic chemical fertilizers.

Plant Growth-Promoting Rhizobacteria Improve Rice Response to Climate Change Conditions.

Rice is one of the most important crops in the world and is considered a strategic crop for food security. Furthermore, the excessive use of chemical fertilizers to obtain high yields causes environmental problems. A sustainable alternative includes taking advantage of beneficial bacteria that promote plant growth. Here, we investigate the effect of five bacterial biofertilizers from halophytes on growth, and we investigate photosynthetic efficiency in rice plants grown under saline conditions (0 and 85 mmol L-1 NaCl) and future climate change scenarios, including increased CO2 concentrations and temperature (400/700 ppm and 25/+4 °C, respectively). Biofertilizers 1-4 increased growth by 9-64% in plants grown with and without salt in both CO2- temperature combinations, although there was no significant positive effect on the net photosynthetic rate of rice plants. In general, biofertilizer 1 was the most effective at 400 ppm CO2 and at 700 ppm CO2 +4 °C in the absence of salt. Inocula 1-5 also stimulated plant length at high CO2 levels without salt. Finally, the positive effect of biofertilization was attenuated in the plants grown under the interaction between salt and high CO2. This highlights the significance of studying biofertilization under stress interaction to establish the real potential of biofertilizers in the context of climate change conditions.

Quantitative Proteomics at Early Stages of the Symbiotic Interaction Between Oryza sativa and Nostoc punctiforme Reveals Novel Proteins Involved in the Symbiotic Crosstalk.

Symbiosis between cyanobacteria and plants is considered pivotal for biological nitrogen deposition in terrestrial ecosystems. Despite extensive knowledge of the ecology of plant-cyanobacterium symbioses, little is known about the molecular mechanisms involved in recognition between partners. Here, we conducted a quantitative sequential window acquisition of all theoretical fragment ion spectra mass spectrometry pipeline to analyze protein changes in Oryza sativa and Nostoc punctiforme during early events of symbiosis. We found differentially expressed proteins in both organisms linked to several biological functions, including signal transduction, adhesion, defense-related proteins and cell wall modification. In N. punctiforme we found increased expression of 62 proteins that have been previously described in other Nostoc-plant symbioses, reinforcing the robustness of our study. Our findings reveal new proteins activated in the early stages of the Nostoc-Oryza symbiosis that might be important for the recognition between the plant and the host. Oryza mutants in genes in the common symbiosis signaling pathway (CSSP) show reduced colonization efficiency, providing first insights on the involvement of the CSSP for the accommodation of N. punctiforme inside the plant cells. This information may have long-term implications for a greater understanding of the symbiotic interaction between Nostoc and land plants.

Conization and lymph node evaluation in low-risk cervical cancer. Is it time to avoid radical surgery? Retrospective series and literature review.

OBJECTIVES: To evaluate the oncologic and obstetric outcomes of patients with low-risk cervical cancer who underwent conization and lymphatic evaluation to preserve fertility. METHODS: Data were collected retrospectively from September 2013 to February 2021. Eligibility criteria included Women with cervical cancer (aged <45 years) who underwent fertility preservation treatment, [stage IA1 with positive lymphovascular space invasion (LVSI), stage IA2, or stage IB1 (≤2 cm) with less <10 mm cervical stromal invasion, according to the International Federaltion of Gynecology and Obstetrics (FIGO) 2018 staging system] aged ≤45 years who wished to preserve their fertility were included in this study. All patients were treated with cervical conization(s) and laparoscopic lymph node evaluation [pelvic lymphadenectomy and/or sentinel lymph node (SLN) mapping]. Oncologic and obstetric outcomes were evaluated. RESULTS: Overall, 31 patients met the inclusion criteria; 15 (48.3%) women were nulliparous. There were 8 IA1LVSI+ (25.8%), 11 IA2 (35.4%) and 12 IB1 (31.7%) tumours, according to 2018 FIGO stage classification. Most patients had squamous cell carcinoma (77.4%). Lymphovascular space involvement was found in thirteen patients (41.9%). Reconization was performed in 17 (54.8%) patients, of which 6(35.2%) were done due to compromised margins, 4(23.5%) for margins under than 3 mm, 3(17.6%) for unreported or coagulated margins and 4(23.5%) because previous conization was done in another institution and we could not obtain the paraffin blocks for pathology review. Twenty patients had MRI and eleven CT scan. Nine (30%) patients had a complete bilateral pelvic lymph node dissection, 9 (26.6%) had SLN mapping with pelvic lymphadenectomy, and 13 (43.3%) had SLN mapping alone after bilateral SLN identification at surgery. After a median follow-up of 41.4 months (range 2-90 months), no recurrences have been detected. In terms of obstetrial outcome, 11 patients attempted pregnancy and 9 became pregnant. First-trimester miscarriage occurred in one patient. Five patients delivered at term by caesarean section, one of them requiring hysterectomy at the time of delivery. Pathology did not show residual disease. Two patients had a vaginal delivery at 38 weeks. One pregnancy is still ongoing. CONCLUSION: Cervical conization with lymph node assessment by SLN mapping/lymphadenectomy is an oncologic safe procedure in patients with low-risk cervical cancer.

Neoadjuvant chemotherapy in early-stage cervical cancer (<2 cm) before conization for fertility preservation: is there any advantage over upfront conization?

BACKGROUND: Neoadjuvant chemotherapy before fertility-sparing surgery is an accepted option for patients with cervical tumors between 2 cm and 4 cm. There is a paucity of data regarding its role in patients with tumors <2 cm. Our objective was to compare the oncological and obstetrical outcomes between patients who underwent neoadjuvant chemotherapy before cervical conization versus upfront cervical conization in patients with cervical cancer with tumors <2 cm. METHODS: We conducted a systematic literature review and searched MEDLINE, EMBASE, and CINAHL (from 1995 to March 2020) using the terms: uterine cervix neoplasms, cervical cancer, fertility-sparing surgery, fertility preservation, conization, cone biopsy, and neoadjuvant chemotherapy. We included manuscripts with information on patients with tumor size <2 cm, lymph node status, follow-up, oncological and obstetrical outcome, and toxicity related to neoadjuvant chemotherapy. We excluded review articles or articles with duplicated patient information. RESULTS: We identified 12 articles, including 579 patients. For final analysis, 261 patients met inclusion criteria. The most common histology was non-squamous cell carcinoma (62%). Median follow-up time was 63.5 (range 7-122) months for the neoadjuvant chemotherapy group and 48 (range 12-184) months for the upfront cervical conization group. There was no difference in either overall survival (neoadjuvant chemotherapy group 100% vs upfront cervical conization 99.7%, p=0.79) or disease-free survival (neoadjuvant chemotherapy 100% vs upfront cervical conization 98.9%, p=0.59) between the groups. Fertility preservation rate was 81.4% versus 99.1% (p<0.001) favoring upfront cervical conization. No statistically significant differences were seen in live birth rate or pregnancy loss. Also, we found that all neoadjuvant chemotherapy patients reported chemotherapy-related toxicity (30.7% grade 3 and 69.2% grade 1-2). CONCLUSIONS: There was no difference in disease-free survival or overall survival between patients who underwent neoadjuvant chemotherapy followed by conization and upfront cervical conization. Patients who underwent upfront cervical conization had a higher fertility preservation rate.

Neoadjuvant chemotherapy prior to fertility-sparing surgery in cervical tumors larger than 2 cm: a systematic review on fertility and oncologic outcomes.

BACKGROUND: Management of cervical cancer tumors >2 cm has been a subject of controversy, with management often considered as either up-front radical trachelectomy or neoadjuvant chemotherapy before fertility-sparing surgery. METHODS: A systematic literature review was conducted following the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) checklist. This study was registered in the International Prospective Register of Systematic Reviews (PROSPERO). We searched Medline through PubMed, EMBASE, Cochrane Central Register of Controlled Trials, SCOPUS, and OVID between January 1985 and December 2020. Eligibility criteria for selecting studies were English language, randomized controlled trials, and observational studies including information on fertility and oncologic outcomes. All titles were managed in EndNote X7. Risk of bias was evaluated using the Joanna Briggs Institute (JBI) critical appraisal checklist for observational studies. RESULTS: Twenty-three studies with 205 patients who received neoadjuvant chemotherapy were included. The majority of patients (92.2%, n=189) had stage IB FIGO 2009 cervical cancer. The preferred regimen used was cisplatin in combination with paclitaxel or ifosfamide (80%, n=164). One hundred and eighty patients (87.8%) underwent fertility-sparing surgery; radical vaginal trachelectomy, abdominal trachelectomy, conization, and simple trachelectomy were performed in 62 (34.4%), 49 (27.2%), 34 (18.9%), and 26 (14.4%) women, respectively. In nine patients (5%) the type of procedure was not specified. The follow-up time reported in all studies ranged between 6 and 69 months. In 112 women who sought a pregnancy after surgery, 84.8% (n=95) achieved a gestation.The global recurrence and death rates were 12.8% and 2.8%, respectively. CONCLUSION: Neoadjuvant chemotherapy followed by fertility-sparing surgery is a promising strategy that might allow fertility preservation in highly selected patients with cervical cancer with tumors >2 cm while providing acceptable oncologic outcomes. Results of prospective studies are required to validate its oncological safety. SYSTEMATIC REVIEW REGISTRATION NUMBER: PROSPERO CRD42020203789.

Emergency Embolization of Pelvic Vessels in Patients With Locally Advanced Cervical Cancer and Massive Vaginal Bleeding: A Case Series in a Latin American Oncological Center.

PURPOSE: Locally advanced cervical cancer may present with uncontrollable vaginal bleeding in up to 70% of cases. Pelvic vessel embolization has been used as an urgent maneuver for achieving fast hemostatic control. This report describes outcomes of selective pelvic vessel embolization in patients with severe bleeding due to a locally advanced cervical cancer. METHODS: In this retrospective study, technical aspects, clinical variables, and bleeding-related morbidity were described. The frequency of recurrent disease and the vital status at 1 year of follow-up were determined. Analysis was performed with statistical software R, version 3.6.2. The setting was Instituto Nacional de Cancerología- Bogotá, Colombia, between January 2009 and July 2017. RESULTS: A total of 47 patients were included. Median age was 44 years (range, 26-70 years). The pre-embolization median hemoglobin level was 7.9 g/dL (range, 5.0-11.3 g/dL). Blood transfusions were administered to 41 women (87.2%). Bleeding control was achieved in 95.7% of cases in the first 24 hours after the embolization. There were no major complications. In 17 cases (36.2%), minor complications were reported; the most common was pelvic pain. In 17.1% of cases, a second embolization was required. After 12 months of follow-up, 27.7% of patients were alive without disease, 44.7% were alive with disease, and 25.5% of them have died of cervical cancer progression. CONCLUSION: Selective pelvic vessel embolization is a useful alternative in patients with locally advanced cervical cancer and life-threatening bleeding. Its impact on recurrent disease and death due to oncologic cause is not clear.

Alternative management for gynecological cancer care during the COVID-2019 pandemic: A Latin American survey.

OBJECTIVE: To determine the acceptance rate of treatment alternatives for women with either preinvasive conditions or gynecologic cancers during the COVID-19 pandemic among Latin American gynecological cancer specialists. METHODS: Twelve experts in gynecological cancer designed an electronic survey, according to recommendations from international societies, using an online platform. The survey included 22 questions on five topics: consultation care, preinvasive cervical pathology, and cervical, ovarian, and endometrial cancer. The questionnaire was distributed to 1052 specialists in 14 Latin American countries. A descriptive analysis was carried out using statistical software. RESULTS: A total of 610 responses were received, for an overall response rate of 58.0%. Respondents favored offering teleconsultation as triage for post-cancer treatment follow-up (94.6%), neoadjuvant chemotherapy in advanced stage epithelial ovarian cancer (95.6%), and total hysterectomy with bilateral salpingo-oophorectomy and defining adjuvant treatment with histopathological features in early stage endometrial cancer (85.4%). Other questions showed agreement rates of over 64%, except for review of pathology results in person and use of upfront concurrent chemoradiation for early stage cervical cancer (disagreement 56.4% and 58.9%, respectively). CONCLUSION: Latin American specialists accepted some alternative management strategies for gynecological cancer care during the COVID-19 pandemic, which may reflect the region's particularities. The COVID-19 pandemic led Latin American specialists to accept alternative management strategies for gynecological cancer care, especially regarding surgical decisions.

Endophytic Colonization of Rice (Oryza sativa L.) by the Symbiotic Strain Nostoc punctiforme PCC 73102.

Cyanobacteria are phototrophic microorganisms able to establish nitrogen-fixing symbiotic associations with representatives of all four of the major phylogenetic divisions of terrestrial plants. Despite increasing knowledge on the beneficial effects of cyanobacteria in rice fields, the information about the interaction between these microorganisms and rice at the molecular and structural levels is still limited. We have used the model nitrogen-fixing cyanobacterium Nostoc punctiforme to promote a long-term stable endophytic association with rice. Inoculation with this strain of hydroponic cultures of rice produces a fast adherence of the cyanobacterium to rice roots. At longer times, cyanobacterial growth in the proximity of the roots increased until reaching a plateau. This latter phase coincides with the intracellular colonization of the root epidermis and exodermis. Structural analysis of the roots revealed that the cyanobacterium use an apoplastic route to colonize the plant cells. Moreover, plant roots inoculated with N. punctiforme show both the presence of heterocysts and nitrogenase activity, resulting in the promotion of plant growth under nitrogen deficiency, thus providing benefits for the plant.

Single-Port/Pneumovagina Technique for Two Further Applications.

OBJECTIVE: To demonstrate a novel technique to surgically treat certain vaginal conditions. DESIGN: Technical video demonstrating 2 cases in which the technique is used. SETTING: Gynecological Minimally Invasive and Robotic Surgery Unit at Clínica Universitaria (private clinic), Concepción, Chile. INTERVENTIONS: Local institutional review board was consulted, and this study was exempted from approval. Institutional ethics committee approved the study and publication of these data. A 35-year-old woman with a bicornuate unicollis uterus presented with dyspareunia. Her examination revealed an incomplete longitudinal vaginal septum. Her right hemivagina was slightly wider than the left one. With the patient under spinal anesthesia, we performed a complete resection of the septum using the single-port/pneumovagina technique (SPPT). A 36-year-old woman who was nulligravida presented with dyspareunia. On clinical exam she had a 3-cm leiomyoma in the proximal vaginal third. Doppler-powered pelvic ultrasound ruled out any vascular communication with the cervix. We performed a vaginal myomectomy using the SPPT under spinal anesthesia. In this particular case we used a fourth trocar in the gel cap to use a myoma screw. With this technique we created a pneumovagina occluding the introitus with the aid of a single-port device (GelPoint Path; Applied Medical, Rancho Santa Margarita, CA). We selected this particular device, designed for transanal surgery, because its access channel avoids gas leakage after applying gentle pressure on the cap. The working cannel is 4 × 4.5 cm, and up to 4 trocars can be inserted in the gel cap. We use 12 mm Hg of pressure to create the pneumovagina and 5 L/min flow to maintain it. Similar approaches have been described for treating eroded and/or infected sacrocolpopexy mesh [1-3]. One could question the utility of this approach over conventional vaginal surgery, and in this sense we believe it provides both the surgeon and surgical assistant a much more comfortable and ergonomic position while performing surgery. It also improves the view of anatomic structures for the surgical team, which in conventional vaginal surgery is limited only to the surgeon. Both procedures were uneventful. The operation time for the first patient was 5 minutes, and the patient was discharged 4 hours later. The operation time for the second patient lasted 35 minutes, and she was discharged 12 hours later. CONCLUSION: The creation of a pneumovagina with the application of a single-port device provides an excellent view of vaginal structures and allows the application of laparoscopic techniques to perform vaginal surgeries in a much more ergonomic fashion compared with conventional vaginal surgery.

Cytochrome c6 is the main respiratory and photosynthetic soluble electron donor in heterocysts of the cyanobacterium Anabaena sp. PCC 7120.

Cytochrome c6 is a soluble electron carrier, present in all known cyanobacteria, that has been replaced by plastocyanin in plants. Despite their high structural differences, both proteins have been reported to be isofunctional in cyanobacteria and green algae, acting as alternative electron carriers from the cytochrome b6-f complex to photosystem I or terminal oxidases. We have investigated the subcellular localization of both cytochrome c6 and plastocyanin in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 grown in the presence of combined nitrogen and under diazotrophic conditions. Our studies conclude that cytochrome c6 is expressed at significant levels in heterocysts, even in the presence of copper, condition in which it is strongly repressed in vegetative cells. However, the copper-dependent regulation of plastocyanin is not altered in heterocysts. In addition, in heterocysts, cytochrome c6 has shown to be the main soluble electron carrier to cytochrome c oxidase-2 in respiration. A cytochrome c6 deletion mutant is unable to grow under diazotrophic conditions in the presence of copper, suggesting that cytochrome c6 plays an essential role in the physiology of heterocysts that cannot be covered by plastocyanin.

Management of enzyme diversity in high-performance cellulolytic cocktails.

BACKGROUND: Modern biorefineries require enzymatic cocktails of improved efficiency to generate fermentable sugars from lignocellulosic biomass. Cellulolytic fungi, among other microorganisms, have demonstrated the highest potential in terms of enzymatic productivity, complexity and efficiency. On the other hand, under cellulolytic-inducing conditions, they often produce a considerable diversity of carbohydrate-active enzymes which allow them to adapt to changing environmental conditions. However, industrial conditions are fixed and adjusted to the optimum of the whole cocktail, resulting in underperformance of individual enzymes. RESULTS: One of these cellulolytic cocktails from Myceliophthora thermophila has been analyzed here by means of LC-MS/MS. Pure GH6 family members detected have been characterized, confirming previous studies, and added to whole cocktails to compare their contribution in the hydrolysis of industrial substrates. Finally, independent deletions of two GH6 family members, as an example of the enzymatic diversity management, led to the development of a strain producing a more efficient cellulolytic cocktail. CONCLUSIONS: These data indicate that the deletion of noncontributive cellulases (here EG VI) can increase the cellulolytic efficiency of the cocktail, validating the management of cellulase diversity as a strategy to obtain improved fungal cellulolytic cocktails.

Cyt c6-3: A New Isoform of Photosynthetic Cyt c6 Exclusive to Heterocyst-Forming Cyanobacteria.

All known cyanobacteria contain Cyt c6, a small soluble electron carrier protein whose main function is to transfer electrons from the Cyt b6f complex to PSI, although it is also involved in respiration. We have previously described a second isoform of this protein, the Cyt c6-like, whose function remains unknown. Here we describe a third isoform of Cyt c6 (here called Cytc6-3), which is only found in heterocyst-forming filamentous cyanobacteria. Cyt c6-3 is expressed in vegetative cells but is specifically repressed in heterocysts cells under diazotrophic growth conditions. Although there is a close structural similarity between Cyt c6-3 and Cyt c6 related to the general protein folding, Cyt c6-3 presents differential electrostatic surface features as compared with Cyt c6, its expression is not copper dependent and has a low reactivity towards PSI. According to the different expression pattern, functional reactivity and structural properties, Cyt c6-3 has to play an as yet to be defined regulatory role related to heterocyst differentiation.

Recalcitrant carbohydrates after enzymatic hydrolysis of pretreated lignocellulosic biomass.

BACKGROUND: To reduce the cost of the enzymes for the hydrolysis of lignocellulosic biomass, two main strategies have been followed: one, the reduction of enzyme dosing by the use of more efficient and stable enzymatic cocktails; another, to include accessory enzymes in the cocktails to increase yields by reducing the recalcitrant carbohydrate fraction remaining at the end of the process. To guide this second strategy, we have explored the chemical bond composition of different fractions of recalcitrant carbohydrates after enzymatic hydrolysis. RESULTS: Two lignocellulosic feedstocks of relevance for the biofuels industry have been analyzed, corn stover and sugarcane straw. On comparing the composition of chemical bonds of the starting pretreated material with samples after standard and forced hydrolysis (with enzyme overdosing), we obtained similar sugar and chemical bond composition. CONCLUSIONS: This suggests that the current enzymatic cocktails bear the set of enzymes needed to hydrolyze these feedstocks. From our point of view, the results show the need for a parallel fine-tuning of the enzymatic cocktails with the pretreatment process to maximize sugar release yield.