Supplemental Digital Content is available in the text Preoperative neoadjuvant chemoradiotherapy (NACR) is used to reduce tumor size for easier resection or improved resectability rates. Considering the difficulties regarding health insurance and health resources in China, an evidence-based short-course neoadjuvant chemoradiotherapy with surgery to cure patients was performed. This study compared the postoperative effects between short-course neoadjuvant chemoradiotherapy and surgery and surgery without neoadjuvant chemoradiotherapy.The current retrospective study was based on a rectal cancer database, including 274 patients diagnosed with rectal cancer between January 2014 and October 2016. Data were analyzed with respect to curative rate, postoperative recovery indicators , chemoradiotherapy-related indicators [white blood cell count (WBC) and carcinoembryonic antigen (CEA) levels], and adverse effects indicators, evaluated according to Common Terminology Criteria for Adverse Events Version 4.0.P > .05) in terms of radical resection rates and the times to urinary catheter removal and passing flatus (P > .05). Statistically significant differences (P < .05) in terms of earlier time for removal of the nasogastric and drainage tubes and time to first oral feeding were observed in the combined therapy group. The decreases in WBC and CEA levels in the combined therapy group were significantly greater than those in the surgery group 1 week after surgery (P < .05); after 1 month, the CEA decrease in the combined therapy group was significantly greater than that in the surgery group (P < .05). More patients in the combined therapy group experienced vomiting, indigestion, dehydration, oral mucositis, sensory neuritis, and alopecia compared with those in the surgery group 1 week after surgery (P < .05); after 1 month, only the incidence of alopecia was higher in the combined therapy group (P < .05).There was no significant difference between the combined therapy and surgery groups (The combined therapy group demonstrated earlier postoperative recovery compared with the surgery group. Short-course neoadjuvant chemoradiotherapy with surgery may lead to postoperative treatment-related adverse effects of varying degrees; however, these adverse effects eventually improve with time. Clinical evidence suggests that neoadjuvant chemoradiotherapy before surgery could effectively reduce the tumor size, which would debase the disease staging as well make resection easier or improve the resectability rate.–10Rectal cancer (RC) remains one of the most common and dangerous gastrointestinal malignant tumors, with a significant economic and health burden in China because of its increasing incidence. recommend neoadjuvant chemoradiotherapy as a long-course treatment consisting of chemotherapy biweekly for at least 2 to 3 months and radiotherapy with a total dose of 45–50 Gy spanning at least 5 weeks. This recommendation presents unique challenges in China, where most patients have to pay for their treatments as there is limited coverage from public or private health insurance.–12 This is further compounded by the uneven health resource distribution between urban and rural areas, as it is common for only large hospitals located in cities to offer high-quality medical care, including chemoradiotherapy. Thus, patients in the low economic strata and rural regions have difficulty completing the recommended NACR, prompting physicians in China to launch a modified regimen shortening the NACR to 5 days to achieve the best benefits for patients with limited resources.The NCCN (2012) guidelines–16 Moreover, no significant statistical difference was reported in local remission rate and long-course survival benefits between long-course and short-course neoadjuvant chemoradiotherapy,–16 even though the rate of adverse events in long-course chemoradiotherapy were higher than those of the short course in RC patients. As a result, the National Institute for Health and Clinical Excellence (NICE) guidelines in England also recommended short-course NACR in patients with locally advanced RC.However, the advantages of short-course neoadjuvant chemoradiotherapy, including shorter treatment times, lower cost, and weaker drug adverse effects, less reportedly impact the quality of patients’ lives. For these patients, professional nurses involved in colorectal surgery need to be aware of the potential clinical problems and implement corresponding management approaches. This study evaluated the impact of short-course neoadjuvant chemoradiotherapy on postoperative recovery in our institution. A clinical team in our colorectal surgery department performed a retrospective controlled study to compare the postoperative effects between short-course NACR and standard surgery without NACR.Adding steps in the new treatment method inevitably leads to additional risks of side effects related to surgery, postoperative recovery, and relative nursing interventions.22.1a.Consented to surgery.b.Diagnosed with colorectal cancer by preoperative colonoscopy and postoperative pathological examination.c.Initial diagnosis of colorectal cancer.d.TNM  stage I or IV cases were excluded.e.Patients that experienced morbidity such skin changes  during chemotherapy or radiotherapy prior to this hospitalization were excluded.f.Patients who received chemotherapy or radiotherapy 1 month after surgery and those who were taking medications with potential adverse effects similar to those of chemotherapeutic agents, including hormones and immunosuppressive drugs, were also excluded.g.We also excluded patients with more than 20% missing data.A total of 274 patients diagnosed with rectal cancer were selected for this retrospective review. These patients were diagnosed in our department between January 2014 and October 2016; the surgery group was enrolled between January 2014 and January 2015, whereas the combined therapy (short-course neoadjuvant chemoradiotherapy and surgery) group was enrolled between October 2015 and October 2016 based on the following criteria (Chart 1): 94 and 104 cases were categorized as Stage II and Stage III disease, respectively. Patient ages ranged from 28 to 80 years and the mean age was 62.6 years. The patients were divided to the combined therapy (82 cases) and surgery (116 cases) groups. Both groups had the same baseline data  and the International Union Against Cancer (UICC),2.2The collected data included hospital admission and 1-month postsurgery clinical follow-up records obtained from the Medical Records department. Our department has a professional staff that liaises with patient groups by regularly conducting telephone follow-ups. The periods included in the study were from admission and 1 month postsurgery, whereas the observation points were before neoadjuvant chemoradiotherapy and 1 week and 1 month after surgery. Data were saved in Excel .2.32  + LV 400 mg/m2  + 5-FU 400 mg/m2 (iv.) and began to receive 5-FU 1200 mg/m2/d .Patients in the combined therapy group consented to under neoadjuvant chemoradiotherapy and subsequent operation after 1 week. This plan included 5 × 5 Gy radiotherapy for 5 consecutive days with a concurrent cycle of FOLFOX at day 2 of radiotherapy. The FOLFOX regimen included oxaliplatin 85 mg/mPatients in the surgery group received only surgery, which was a laparotomy procedure, including corrective operation and a palliative procedure (treatments not performed to resect or debulk the tumor).2.4 and included:1.Gastrointestinal reactions: nausea , vomiting, indigestion , diarrhea, and gastroesophageal reflux (heartburn and indigestion).2.Rectum-related phenomena: intestinal obstruction, bleeding in the enteric cavity, intestinal ulceration/perforation, and intestinal stenosis.3.Fever, oral mucositis, acne-like rashes, sensory neuritis, and alopecia.We evaluated the effect of neoadjuvant chemoradiotherapy on treatment and patient recovery by comparing curative rates. The curative rate was defined as the number of radical resection cases divided by the total number of cases, multiplied by 100%. The postoperative recovery indicators include the times to remove the nasogastric tube, urinary catheter, and drainage tube and the times to first oral feeding and first passing of flatus postsurgery. The chemoradiotherapy-related indicators included white blood cell (WBC) count and carcinoembryonic antigen (CEA) level. We compared the last available data before the administration of NACR to the values measured 1 week or 1 month after surgery. The data values before NACR were subtracted from the postsurgery measurements. The adverse effects indicators were developed according to Common Terminology Criteria for Adverse Events Version 4.0 (CTCAE v4.0)2.5We categorized the adverse effects into 5 degrees according to CTCAE v4.0: first-degree (low level) was defined as zero or a few symptoms diagnosed in the clinic and that required no intervention. Second-degree (medium level) were defined as diseases requiring local, small, noninvasive therapy or in which the instrumental activities of daily living according to age were limited. Third-degree was defined as the presence of indicators that could lead to hospitalization, lengthen the recovery time, or result in disabilities that were serious or had clinically significant impacts that did not threaten life or limit the personal activities of daily living. Fourth-degree was defined as life-threatening adverse effects that required emergency treatment. Finally, fifth-degree adverse events were defined as death related to adverse effects.2.6To control for bias, we optimized the schedule before starting the study and screened clinical cases and follow-up data in the light of the study inclusion and exclusion criteria. Two people logged data and professional statisticians analyzed data; all were blinded during the logging and analysis.t tests; otherwise, U tests were used instead. χ2 or Fisher exact tests were used for enumeration data, while Wilcoxon rank-sum tests were used for ranked data based on an α = .05.The data were processed using SPSS Statistics for Windows, version 17.0 . Consecutive variable data with equal variance were assessed using 3P > .05) groups in terms of radical resection rates. Among postoperative indicators, there were also no significant differences between groups in the times to urinary catheter removal and passing flatus (P > .05). A statistically significant difference (P < .05) was observed in the earlier removal times for nasogastric and drainage tubes and the time to first oral feeding in the combined therapy group  × 109/L before neoadjuvant chemoradiotherapy and (3.0 ± 2.8) × 109/L and (4.4 ± 3.9) × 109/L in the combined therapy and surgery groups, respectively, 1 week after surgery. The WBC decrease was significantly higher in the combined therapy group than that in the surgery group (P < .05). The CEA values of patients in the combined therapy and surgery groups were 21.4 ± 25.1 and 26.5 ± 30.7 ng/mL before NACR and 13.8 ± 18.3 and 18.8 ± 21.0 ng/mL, respectively, 1 week after surgery. The decrease in CEA value in the combined therapy group was significantly higher than that in the surgery group (P < .05)  × 10.1 × 109/P > .05). More patients in the combined therapy group experienced vomiting, indigestion, dehydration, oral mucositis, sensory neuritis, and alopecia compared with those in the surgery group (P < .05)  Table .9/L and 6.1 ± 2.5 × 109/L before neoadjuvant chemoradiotherapy and 4.8 ± 3.5 × 109/L and 4.7 ± 3.9 × 109/L at 1 month after surgery. The difference in WBC decrease between the groups was not statistically significant (P > .05). The CEA values in the combined therapy and surgery groups were 21.4 ± 25.1 and 26.5 ± 30.7 ng/mL before neoadjuvant chemoradiotherapy and 9.5 ± 9.3 and 19.0 ± 20.7 ng/mL 1 month after surgery. The decrease in CEA value in the combined therapy was significantly higher than that in the surgery group (P < .05) . Alopecia was significantly more common in the combined therapy group compared with the surgery group (P < .05)  Table .4–5 There is mounting evidence of the advantages of preoperative neoadjuvant chemoradiotherapy from a number of randomized controlled trials,,22–28 leading colorectal surgeons, including those in China, to adopt the practice. A number of neoadjuvant chemoradiotherapy treatment practices were developed in large domestic hospitals, offering patients the benefits described in international scientific papers.–33 Easing the economic burden for patients requiring combined therapy was the most crucial reason that this study was undertaken. The combined therapy group in our study demonstrated earlier postoperative recovery than that in the surgery group, thus proving its advantage. Excellent postoperative recovery depends on the reduction in cancer size because of neoadjuvant chemoradiotherapy, which may minimize the surgical risk and difficulty, shorten surgical time, and reduce damage caused by the operation.–10 Although the retrospective data did not include the tumor stage and size before surgery, we hope these data will be included as a research point in future prospective studies.The combination of neoadjuvant chemoradiotherapy and surgery is widely acknowledged in international guidelines as a regimen for colorectal cancer treatment.–36 It is thus imperative to be aware of adverse event to be prepared to provide an optimal response during the perioperative period to enhance the comprehensive quality when implementing a colorectal treatment plan based on neoadjuvant chemoradiotherapy. Acute adverse effects  are observed in the first few hours after chemoradiotherapy, whereas delayed adverse effects  are seen 7 to 9 days after surgery or even later. This period lasted 1 or 2 months or longer in long-course neoadjuvant chemoradiotherapy ; however, the preoperative treatment-related adverse effects caused by long-course neoadjuvant chemoradiotherapy were harder to assess. None have been documented so far. Our study aimed to explore the possibility of regulating the adverse effects of short-course neoadjuvant chemoradiotherapy during the perioperative and late recovery periods. Our research came from a single-treatment group, which utilized a computer algorithm recovery mode of fast-track surgery.–39 The combined therapy and surgery groups managed with fast-track surgery both showed good postoperative recovery indicators; however, adverse effects related to radiotherapy were reported in the combined therapy group.Short-course neoadjuvant chemoradiotherapy may lead to late adverse effects after surgery. Clinical staffs were concerned that the adverse effects caused by the combined therapy would limit satisfaction with hospitalization and compliance with long-course treatment. Neoadjuvant chemoradiotherapy may lead to immune dysfunction, nausea, vomiting, rashes, alopecia, neurodermatitis, and hepatotoxicity.–41 Changes in WBC made clinical sense as an index to evaluate the adverse effects of vein radiotherapy. Patients who received radiotherapy often had experienced decreased leukocyte counts even though leukocyte crisis was the result of malignant myelosuppression. In our study, the WBC values in the combined therapy group were less than those in the surgery group, suggesting that the effects of combined therapy were worse before neoadjuvant chemoradiotherapy or 1 week after surgery. The WBC did not differ significantly between the combined therapy and surgery groups and the drop in CEA in the combined therapy group was significantly less than that in the surgery group 1 month after surgery. Therefore, patients in the recent recovery period may have low WBC levels, which could be an important factor affecting postoperative recovery. Developing a postsurgery recovery plan and discharging at a reasonable time could help to avoid these risks and guarantee good short-course results for combined therapy of malignant tumor. Close monitoring for leukocyte crisis and promptly administering relevant treatment may reduce the risk of infection in patients after surgery.The adverse effects of radiotherapy disappeared over time.Although the combined therapy group had an increased probability of developing adverse effects one week after surgery, specifically adverse effects related to vein radiotherapy, adverse effects such as vomiting, oral mucositis, indigestion, sensory neuritis, and alopecia improved and eventually disappeared over time, with no significant differences observed between the groups after 1 month. However, because of its characteristics, alopecia cannot be corrected during this short period; therefore, patients who consent to this procedure must be made aware of this. In general, the adverse effects experienced by patients were generally mild and disappeared over time. Patients with severe adverse effects (third degree or more) included 2 patients with diarrhea and another 2 patients with dehydration 1 week after surgery. The series of problems may be resolved and improved with long-course postoperative follow-up and treatment compliance of patients if a reasonable postoperative short-course recovery plan was arranged beforehand.Several limitations of this study should be noted. First, this retrospective study was limited in scope to preliminarily test the hypothesis. Future prospective randomized controlled trials are necessary to confirm these findings. Second, long-course chemoradiotherapy is also administered to rectal cancer patients in this clinic; however, we did not compare patients who had received long-course and short-course courses. Therefore, further research is necessary to compare these treatments. Lastly, the 3 and 5-year survival rates were not recorded.In summary, combining neoadjuvant chemoradiotherapy with surgery may cause postoperative treatment-related adverse effects to varying degrees, notably leucocyte crisis and peripheral neuritis; however, these adverse effects improved with time postsurgery. Furthermore, these adverse effects did not affect the postoperative recovery time.


Results are verified against a detailed theoretical treatment, which also describes how the system can be scaled up to a universal quantum hyperpolarisation platform for macroscopic samples. Our results demonstrate the prospects for this approach to nuclear spin hyperpolarisation for molecular imaging and spectroscopy and its potential to extend beyond into other scientific areas.Hyperpolarisation of nuclear spins is important in overcoming sensitivity and resolution limitations of magnetic resonance imaging and nuclear magnetic resonance spectroscopy. Current hyperpolarisation techniques require high magnetic fields, low temperatures, or catalysts. Alternatively, the emergence of room temperature spin qubits has opened new pathways to achieve direct nuclear spin hyperpolarisation. Employing a microwave-free cross-relaxation induced polarisation protocol applied to a nitrogen vacancy qubit, we demonstrate quantum probe hyperpolarisation of external molecular nuclear spins to ~50% under ambient conditions, showing a single qubit increasing the polarisation of ~10 Molecules with ‘hyperpolarised’ nuclear spins can be used to improve MRI performance but require an efficient polarisation method. Broadway et al. demonstrate a quantum control protocol using a nitrogen vacancy centre inside a diamond to hyperpolarise protons within molecules deposited on the surface. Hyperpolarisation offers enormous gains in signal and spatial resolution which may ultimately lead to the development of molecular MRI and NMR3, with the prospect of revolutionising many areas of research and clinical applications. Well-known hyperpolarisation techniques include high-field/low temperature brute-force methods4, dynamic nuclear polarisation6, optical pumping5, and parahydrogen-induced polarisation7. On the other hand, the rapid advances in semiconductor spin qubit technology for quantum computing10 and quantum sensing14 has opened up the exciting possibility of hyperpolarising nuclear spins at a fundamental level via quantum mechanical protocols24. Despite early progress26, the application of quantum probe technology to this problem still faces a number of significant challenges. To be of practical use, a quantum probe must be capable of polarising a relatively large number of remote nuclear spins external to the probe substrate, ideally under ambient conditions.The hyperpolarisation of nuclear spins within target molecules is a critical and complex challenge in magnetic resonance imaging (MRI)In the following, we address these challenges using a quantum spin probe in diamond as an entropy pump, and, using a combined experimental and theoretical analysis, demonstrate polarisation of external molecular spin ensembles to about 50% over relatively large volumes at room temperature, with the prospect of scaling up to a universal hyperpolarisation platform suitable for clinical applications. In contrast to existing methods, our quantum polarisation approach is tunable to a range of nuclear species, is inherently free of radiofrequency (RF) or microwave fields and extraneous chemistry, and is carried out at room temperature.11. Existing approaches to NV-based hyperpolarisation involve the use of an external microwave field to tune the NV Rabi frequency to the Larmor frequency of the target spins, thereby enabling rotating-frame cross-relaxation via the Az,x and Az,y components of their hyperfine interaction. Our approach, on the other hand, takes advantage of the greater  NV-target hyperfine interaction associated with direct lab-frame cross-relaxation, which allows for an order of magnitude greater target polarisation rate. This is achieved by using an external magnetic field, B, to tune the ground-state spin transition frequency of the NV (ωNV) into resonance with target nuclear spins (ωn)  ≈ 2D/(γNV − γn)27, where γn, γNV are the target and NV gyromagnetic ratios, and D is the NV zero-field splitting . The transfer of magnetisation caused by this interaction thus polarises the target spins into their π-pulse  quantum spin probe in a diamond substrate Fig. 11. Existωn) Fig. . For a ging Fig. . Entropying Fig. , whereinate Fig. . For thelse Fig. .Fig. 1QuR (relative to the NV) and time t to be P; with the evolution of P described byP = 0; where u(R) = A2(R)/2Γ2 is the effective cooling coefficient resulting from the hyperfine coupling A(R) with the NV spin, Γ2 is the dephasing rate of the NV spin β is the effective polarisation diffusion coefficient related to the intra-target interactions, and ΓSL is the spin-lattice relaxation rate of the target spin ensemble. This formulation allows us to predict and describe the spatial extent of polarisation for a given target sample of arbitrary geometry.To quantitatively understand the effect of the CRIP protocol on the target spin ensemble, we developed a microscopic theory that explicitly includes the dipole interactions of ensemble spins and their interaction with a single NV quantum probe  from the NVrum Fig. , and canrum Fig. .13C spin ensemble surrounding a NV probe in the diamond substrate by tuning to the 13C resonant condition at B*(13C) = 1024.9 G. Comparison of the cross-relaxation spectra for CRIP and depolarisation sequences  to calculate the time dependence of the radial polarisation profile for total polarisation times of 1–106 s, as depicted in Fig. P, to the cross-relaxation rate, ΓCR, via Eq. , PMMA, was applied directly to a diamond surface12 with single NV spin probes located 8–12 nm below the surface with an average density of 108 cm−2. The initial criteria for finding suitable NVs involved finding a trade-off between maximising the NV-H coupling , and minimising the coupling to sparse electronic surface defects (deep) which limit the NV linewidth and the background T1 time. The surface is also known to retain a 1–2 nm thick layer of protons of density commensurate with that of PMMA15, which we therefore regard as being part of the target proton system to be polarised.With the basic protocol established, we now move to the polarisation of molecular B*(1H) = 1026.2 G. With a much higher diffusion constant and spin-lattice relaxation rate 16 relative to the intrinsic 13C case, the 1H system effectively reaches steady-state within a few seconds. Application of the CRIP sequence to NV1  will allow for more precise tuning to different nuclear spin species. As the protocol is all optical, scaling up for high-volume production could be achieved by stacking multiple NV arrays , a well-known MRI contrast agent32. Using a single hyperpolarisation cell comprised of two NV arrays in diamond membranes separated by 1 μm , the rate of polarisation transfer to a concentrated 1 M precursor HEP solution is 4 μL s−1 at a polarisation level of 80%. The polarisation levels for different contrast agents in 1 M precursor solutions are plotted against polarisation time  in Fig. −1 for clinical applications34 are achievable.The results presented here indicate that the CRIP protocol could produce macroscopic quantities of MRI contrast agents with high polarisation levels. For example, we consider In summary, we have experimentally demonstrated hyperpolarisation of molecular nuclear spins under ambient conditions by employing a quantum spin probe entropy pump. The technique works at low magnetic fields and room temperature conditions, requires no RF or microwave fields, and operates directly on the target molecules without the need for catalysts or free radicals. With high polarisation rates and tunability, there are excellent prospects for scale-up of the system to produce macroscopic quantities of a range of contrast agents at polarisation levels required for molecular MRI/NMR. The technique can be extended to other nuclear spin species and may also provide pathways in quantum information for initialisation of quantum simulators, or increasing the fidelity of operations through spin-bath neutralisation. Future work will involve external verification of nuclear spin polarisation using conventional NMR techniques.29. In summary, the excitation source is a solid-state laser emitting at a wavelength λ = 532 nm (Laser Quantum Gem 532). The objective lens  is mounted on an XYZ scanning stage (PI P-611.3 NanoCube) to allow fast laser scanning. The PL emitted by the diamond sample is separated from the laser light using a dichroic beam splitter and a band-pass filter, and coupled into a multimode fibre connected to a single photon counting module (Excelitas SPCM-AQRH-14-FC). For T1 measurements, the laser beam is modulated by an acousto-optic modulator  in a double pass configuration, and the PL signal is analysed by a time digitiser (FastComTec P7889). For optically detected magnetic resonance (ODMR) measurements, a 20-μm copper wire is spanned on the surface of the diamond and connected to the output of a microwave generator (Agilent N5181A) modulated by a switch (Mini-Circuits ZASWA-2-50DR+). Laser and microwave modulations are controlled by a programmable pulse generator (SpinCore PulseBlasterESR-PRO 500 MHz). The magnetic field direction and strength were varied by using a permanent magnet affixed to a set of three linear translation stages (PI M-511) allowing XYZ position control. These stages had a resolution of 100 nm which is sufficient to tune the NV into resonance and align along the field along the NV axis, thus avoiding any misalignment issues27.The experimental apparatus consists of a custom-built confocal microscope and a permanent magnet mounted on a scanning stage, the same setup used, and described, in13C measurements was a 1H measurements is a 15N and 14N at 3 keV at a density of 5 × 108 cm−2 each. The sample was annealed at 950 °C for 2 h and was exposed to a soft O2 plasma for 1 minute36. The PMMA was baked onto the surface of the diamond by a heat gun at 85° for 40 min. The diamond has a range of near-surface NV depths (~3–13 nm) determined by NV-NMR spectroscopy using the dynamical decoupling method35.The sample #132) used for the  used for38. When doing this near the GSLAC, we estimate an alignment to less than 0.1°. The magnet was then stepped along the NV direction to vary the magnetic field strength B0. For each magnet position, an ODMR spectrum was recorded for about 1 minute from which we extract ωNV via a Lorentzian fit, before the CRIP sequence is applied. The latter consists of a series of 3-μs laser pulses, sufficient to completely initialise the NV spin state, separated by a wait time τ. The signal plotted in Figs. −1 pulse sequences, which acts to prevent polarisation build up in either direction. The CRIP−1 simply adds a radiofrequency (RF) pulse to flip the NV spin from |0〉 to |−1〉. This π pulse was applied 1 μs after the end of the laser pulse, was 300 ns in duration, and was followed by a wait time τ identical to that used in the preceding CRIP sequence. With the interleaved sequence, there are two independent PL readout, one during the laser pulse following the CRIP sequence, the other following the CRIP−1 sequence. Note that the interleaved sequence continuously polarises the bath but each pulse flips the direction of this polarisation. As such, because of pulse imperfections it may still partly polarise nearby spins.The spectra shown in Figs. T1 measurements presented in main text Figs. τ was continuously swept. To monitor magnetic field drifts caused by thermal fluctuations, we recorded an ODMR spectrum at regular intervals. We can thus ensure that the NV remains on resonance with the target transition, within the NV linewidth  = 1 − 2p, we obtain Equation , since Γ2 defines the width of the cross-relaxation resonances28. We note that Γ2 is expected to decrease upon polarisation of the 13C bath. However, the effect is relatively small . For the 1H case, the dephasing is dominated by surface effects, and is therefore unaffected by the 1H polarisation.Experimentally, ΓP, we may determine the total number of spins polarised at time t:Upon solution of Eq. The data that support the findings of this study are available from the corresponding author upon request.Supplementary Information


These studies showcase the rational design of a family of amyloid-binding imaging agents that could be integrated with new optical approaches for the clinical diagnosis of amyloidoses, where accurate identification of the specific neurodegenerative disease could aid in the selection of a proper course for treatment.The deposition of insoluble protein aggregates in the brain is a hallmark of many neurodegenerative diseases. While their exact role in neurodegeneration remains unclear, the presence of these amyloid deposits often precedes clinical symptoms. As a result, recent progress in imaging methods that utilize amyloid-specific small molecule probes have become a promising avenue for antemortem disease diagnosis. Here, we present a series of amino-aryl cyanoacrylate (AACA) fluorophores that show a turn-on fluorescence signal upon binding to amyloids in solution and in tissue. Using a theoretical model for environmental sensitivity of fluorescence together with  While no cure currently exists for these diseases, progress has been made toward the treatment of late-stage symptoms such as memory loss and restlessness8. Substantial clinical evidence suggests that early diagnosis and intervention may provide the best chance for improving quality of life11. Unfortunately, the differential diagnosis between the various neurodegenerative disorders remains a difficult challenge, particularly during the early stages. For instance, clinical diagnosis of Alzheimer’s disease (AD) can only be tentatively made in late stages by combining neuropsychological evaluations and scanning technologies such as positron emission tomography, while definitive diagnosis is only confirmed from post-mortem examination of the brain12. Diagnostic criteria for other neurodegenerative diseases such as prion-related Creutzfeldt Jakob disease (CJD) are even less well-defined16. Thus, methods that can help diagnose and differentiate patients with neurodegenerative disorders, ideally pre-symptomatically, would be of great clinical value.Progressive neurodegenerative diseases are one of the leading causes of death in the US18. While it is uncertain whether these amyloid deposits are causative of neurodegeneration or are a byproduct of the disease, it is widely accepted that their appearance in the brain precedes clinical symptoms by years, if not decades21. Although the general cross β-sheet organization of amyloids causes their macroscopic structural features to be very similar to one another, Nilsson, Hammarström, and co-workers have reported that conformation-sensitive fluorescent oligothiophenes can distinguish subtle differences in conformation of different strains of prions deposits in vitro23. However, at a molecular level, amyloids associated with different diseases are generally formed from different amyloidogenic proteins with distinct differences in their primary amino acid sequences25. Such molecular differences in composition could potentially be exploited for the differential diagnosis of various neurodegenerative diseases at pre-clinical stages where behavioral and neuropsychological criteria may be unreliable27.One of the defining characteristics of many neurodegenerative diseases is the presence of extracellular or intracellular deposits of misfolded protein aggregates known as amyloids30. These probes are comprised of an electron donor conjugated to an electron acceptor, leading to an enhancement of the fluorescence emission upon binding to an amyloid32. For instance, we showed that an aminonaphthyl 2-cyanoacrylate (ANCA)-based probe (compound 2) was capable of fluorescently labeling amyloid plaques associated with AD or prion disease in neuronal tissue with excellent specificity and reproducibility33. Intriguingly, upon binding to these amyloids, probe 2 exhibited a different color of fluorescence emission when bound to amyloids of different protein compositions34.We previously reported the design of amyloid-binding probes based on a fluorescent molecular rotor motif2 as a spectroscopic ruler. We found that, indeed, probe 2 can differentiate between many of these amyloids, with the measured permittivity values indicating that they all generally contain molecular binding pockets that are relatively non-polar. In an effort to increase the environmental sensitivity of such fluorescent probes across such narrow range of permittivities, we synthesized and evaluated a set of amino-aryl cyanoacrylates (AACAs) with modifications in the length and polarizability of the fluorophores. Ab initio computational studies of probe solvation provided a theoretical molecular interpretation for the effects of different structural features on the difference in dipole moments between the excited and ground states of the probes, as well as provided estimates for the Onsager radius and torsional strain for each fluorophore35. Finally, we evaluated the capability of these new AACA probes to discriminate between β-amyloid (Aβ) and prion plaques in tissue and demonstrate proof-of-concept for using multiple probes to discriminate between amyloids with similar binding pocket polarities in a fluorescence fingerprint assay. These results, together with recent reports on the presence of amyloid deposits in optically accessible tissue such as in the eyes of human patients, suggest an exciting opportunity to develop non-invasive optical tools that allow detection and identification of different amyloidogenic proteins and their respective neurodegenerative diseases44.Inspired by these initial findings, here we measured the permittivity value of several amyloids using probe 2 adopt different conformations upon binding to amyloids due to the influence of different amino acid residues in the β-sheet region46. We theorized that differences in amino acid sequences of amyloidogenic proteins could lead to variations in the polarity of the microenvironment within the binding pocket and, thus, affect the fluorescence properties of environmentally sensitive probes such as 2. With this in mind, we used probe 2 as a spectroscopic ruler to estimate the range of polar environments found in the binding pockets of 11 different proteins/peptides in amyloid form. This set of amyloid samples included natural tissue deposits as well as synthetic or recombinant peptide and protein aggregates in solution  and systemic diseases , in addition to natural bacterial  and seminal amyloids .Molecular dynamics simulations have indicated that amyloid-binding probes such as thioflavin T (ThT) and probe max) of probe 2 when bound to 11 different amyloid species in tissue or in solution is provided in Fig. max, Fig. 2 bound to these amyloids spanned ~40 nm. In order to relate the observed color of fluorescence emission from probe 2 to an intrinsic property of the amyloid binding pocket, we approximated that its environmental sensitivity can be described by the theoretical model introduced by Lippert and Mataga49. This model provides a quantitative approximation of the relationship between the observed Stokes Shift (in wavenumbers), ∆ε, and the refractive index, n, as shown in equation (μe − μg), and the Onsager radius, a, are properties inherent to the fluorophore, h and c are Planck’s constant and the speed of light, respectively, and C is the observed Stokes shift of the fluorophore in a non-solvatochromic environment49.The fluorescence emission  of probe 2 and the single variable of environmental permittivity, ε, by measurement of fluorescence in different solvents with known permittivity  values of bound compound 2  dipole. Relaxation from this state can occur via a combination of fluorescence emission and non-fluorescent mechanical de-excitation . The latter pathway decreases upon restricted rotation due to amyloid binding leading to a significant increase of the emission quantum yield. On the other hand, the emission wavelength depends on the energy difference between the ground and excited state dipoles that, in turn, can be affected by the polarity of the microenvironment. Based on the Lippert-Mataga model, the inherent sensitivity of a fluorophore to polarity is governed by a molecular term , as well as the Onsager cavity radius of solvation, a, occupied by the fluorophore in the solvation field.Central to the fluorescent molecular rotor motif is the electronic coupling of an electron donor with an electron acceptor group through a π-conjugated network. In the specific structure of probe d state S dipole. 1–5 we varied the number of aryl groups between the electron donor and acceptor , in order to examine the effects of the π-network length on environmental sensitivity within the range of local environments available in the binding pockets of amyloids. In compounds 3–5 we altered the heteroaromatic ring in order to evaluate the effect of electron donating heteroatoms on μe, μg and a . To this end, we synthesized and examined the fluorescence properties of a set of five related fluorophores 50:The absorption and fluorescence emission spectra of AACA probes 1–5 as a function of the solvent orientation polarizability . On the other hand, the naphthalene-containing probe (compound 2) was found to have a comparable sensitivity to the naphthalenyl-furan (probe 4) and the naphthalenyl-thiophene (probe 5) probe. We attributed this finding to the fact that while the difference in dipole moments between excited and ground states are expected to be larger for longer probes 4 and 5 compared to probe 2, the increased length of 4 and 5 also increases their Onsager radii compared to compound 2. This result highlights the effect of increasing length of the π-network in AACA probes on increasing both the excited and ground state dipole moments and radius of the fluorogenic core, which have opposing consequences on environmental sensitivity according to equations (3) had the greatest environmental sensitivity, suggesting that the effects of the π-network on the length of the dipole moment and the Onsager radius may not be the only structural features to consider for developing environmentally sensitive fluorescent probes. We, therefore, sought to gain additional molecular insight into the relationship between structure and environmental sensitivity of probes 1–5 using computational techniques.We observed that probe ted Fig. . This obcompound  was foun3 contains the strongest environmental sensitivity . To gain further insight into this observation, we performed density function theory (DFT) calculations of probes 1–5 in vacuum and solvated in various aprotic solvents 2] as a function of the solvent dielectric constant  had the greatest change in dipole strength [(μe − μg)2] compared to probes 1 and 2 as solvent polarity  increased. In contrast to the experimental data for solvatochromism shown in Fig. 5) is expected to produce the greatest difference in excited/ground state dipole moments at large environment polarity. Thus, in theory, probe 5 would be expected to exhibit the greatest environmental sensitivity of all probes tested according to equation (μe − μg)2 rapidly decreases for all probes 1–5 as the surrounding dielectric environment approaches zero . As a result, in low dielectric environments, we would not expect large differences in environment sensitivity for probes 3–5 based on differences in excited/ground state dipole moments alone. Additional DFT calculations determined the radius of probe 3 to be 8.59 Å, compared to 9.01 Å for probe 4 and 9.23 Å for probe 5. Considering equation  states31. The TICT state is known to be stabilized by highly polar environments51. We hypothesized that the enhanced environmental sensitivity of the naphthalenyl-pyrrole probe 3 could arise from torsional strain along the presumed freely rotating single bonds between the electron donor and acceptor of the AACA probes. DFT calculations provided an estimate for the average dihedral angle in the ground and excited states for compounds 3–5 between the naphthalene and the heterocyclic aromatic ring in various dielectric environments , which can further stabilize the excited state and enhance environmental sensitivity46.In the ground state, probes 1–5 could serve as diagnostic tools for amyloid deposition in tissue, we examined the cellular toxicity of the probes in SH-SY5Y neuroblastoma cells. After 24 hours of dosing, we did not observe toxicity at concentrations up to 200 µM for any of the probes compared to control cells  peptides at various time points during the aggregation process, which contained different mixtures of soluble aggregates  that changed over the course of aggregation  of amyloidogenic proteins and peptides. In an initial study towards this goal, we did not observe a significant shift in the fluorescence emission λ1–5 could discriminate amyloids of different disease origin in brain samples, we incubated brain tissue slices from a transgenic (Tg) mouse model for Alzheimer’s disease (19959 PRNP-APPswe/Ind) and from mice inoculated with infectious mouse-adapted chronic wasting disease (mCWD) prions with each probe 1–5 57. True-color microscopy of the resulting stained tissues revealed that each compound bound specifically to amyloid deposits from both types of brain samples . The emission profiles for probes 1–3 bound to Aβ diffuse and dense-core deposits in the hippocampus of Tg AD mice were blue-shifted compared to the same probes bound to prion aggregates in the corpus callosum of the prion-inoculated mice. Emission spectra of the stained amyloids  protein composition, thereby complementing the protein identity-distinguishing capabilities of probes 1–360.Closer inspection of dense-core plaques stained with probes 1–3 to fluorescently discriminate Aβ or prion deposits in neural tissue, Fig. 62.Despite the capability of probes 1, 2, and 5 in various solvents and evaluated their fluorescence properties when bound to Aβ or prion deposits in tissue and insulin or α-synuclein aggregates in solution63. Figure To demonstrate proof-of-concept, we generated calibration curves  can be used to identify living Alzheimer’s patients by fluorescently labeling amyloid-containing deposits in the eyes64, demonstrating one possible translational opportunity for fluorescent amyloid-binding probes in clinical diagnosis. Fluorophores that can discriminate amyloids and identify disease origin may provide additional advantages for such novel optical platforms for diagnosis of specific amyloid-based diseases66.While this work demonstrates that the relative permittivity of the binding pocket of an amyloid is an intrinsic property that can potentially be exploited for identification of the amyloidogenic protein, in many instances the permittivities (which reflect the polar microenvironment within the binding pockets of amyloids) are similar between amyloids of different composition and disease origin. This challenge of identifying the protein content within each amyloid can be overcome by using a fluorescence fingerprint assay, where using multiple AACA probes offers additional capabilities to spectroscopically differentiate such amyloids with similar permittivities. Recent results from a phase II clinical study has already demonstrated that AACA probe 2Cl2) were obtained by passing commercially available pre-dried, oxygen-free formulations through activated alumina columns. Flash column chromatography was performed on silica gel  using Hexanes-EtOAc or EtOAc-MeOH or toluene-acetone or diethyl ether mixtures of increasing polarity. The progress of all the reactions was monitored by thin-layer chromatography (TLC) using glass plates precoated with silica gel-60 F254 to a thickness of 0.5 mm (Merck), and compounds were visualized by irradiation with UV light and/or by treatment with a solution of CAM stain or potassium permanganate (KMnO4) in water stain followed by heating. 13C NMR and 1H NMR spectra were recorded on either 400 MHz or 500 MHz Varian instrument or 500 MHz JEOL instrument. CDCl3 was treated with anhydrous K2CO3, chemical shifts (δ) are quoted in parts per million (ppm) referenced to the appropriate residual solvent peak reference (CDCl3), with the abbreviations s, br s, d, t, q, m, td, dt and qd denoting singlet, broad singlet, doublet, triplet, quartet, multiplet, quartet of doublets, triplet of doublets, doublet of triplets and quartet of doublets respectively. J = coupling constants given in Hertz (Hz). High-resolution Mass spectra (HRMS) were recorded on a trisector WG AutoSpecQ spectrometer. Solvent permittivity values  for solvatochromism fluorescence assays were obtained from Sigma and CRC Press68.Unless otherwise indicated, all commercially available chemical reagents and anhydrous solvents were purchased at the highest commercial quality and were used as received without further purification. All non-aqueous reactions were carried out under argon atmosphere using dry glassware that had been flame-dried under a stream of argon unless otherwise noted. Anhydrous tetrahydrofuran (THF) and dichloromethane (CH1–5.The supplementary information describes the details for the synthesis of AACA probes Sc) deposits, and serum amyloid A (SAA) deposits, and human brain tissue containing Aβ senile plaques were generously provided by Professor C. Sigurdson and colleagues (UCSD School of Medicine). Human patient samples were obtained from the UCSD Alzheimer’s Disease Research Center. Institutional board review was obtained from the UCSD Human Research Protections Program in accordance with the Health Insurance Portability and Accountability Act. Synthetic β-amyloid (1–42) was purchased from PL Laboratories  as a lyophilized powder and stored in an anhydrous environment at −80 °C until use. Full length recombinant α-synuclein was generously provided by Professor D. Selkoe  as a frozen stock in 1xPBS, stored at −80 °C. Recombinant human insulin was purchased from Sigma Aldrich . Fibrillized amyloid aggregates of the seminal peptides SEVI  or semen derived enhancer of viral infection), PAP(85–120), and semenogelin-1 (SEM-1(49–107)), and the bacterial peptide fragments protegrin-1 (PG-1), and curli fragments CsgA-R1 and CsgA-R5 were graciously provided by Professor S. Dewhurst and coworkers from the University of Rochester Medical Center. Samples were stored frozen in 1xPBS or deionized H2O at −80 °C.Tissue samples of mice brain tissue with β-amyloid (Aβ) plaques, prion , and serum amyloid A (SAA), and fibrillized aggregates of insulin, Aβ(1–42), α-synuclein, protegrin-1 (PG-1), curli (CsgA-R1 and CsgA-R5), semen derived enhancer of viral infection ) and related fragment (PAP(85–150), and SEM-1 (semenogelin-1) were then stained or bound with ANCA probe 233. Fluorescence emission wavelengths at the maximum intensity, λmax, were converted to permittivities using equation  and equation , respect and equa2O), anisole, and toluene to a final concentration of 1.5 μM. Excitation and fluorescence emission spectra of each solution were obtained on a PTI spectrofluorimeter under argon in 1 nm increments from 350–520 nm and 400–650 nm, respectively. The wavelength corresponding to the maximum intensity was taken as λmax. For absorption studies, compound solutions in anhydrous solvents under argon were prepared at 5 μM. Absorption spectra were obtained from a NanoDrop 2000 UV-Vis spectrophotometer  using a clear quartz cuvette. Because equation , acetone, dichloromethane (DCM), tetrahydrofuran (THF), diethyl ether EtO, anisol−8 convergence criterion for the density matrix elements, using the hybrid-meta GGA functional M062X70. The fully optimized contracted triple-ζ all electron Gaussian basis set developed by Ahlrichs and co-workers was employed for all the elements71. The studied systems have been fully optimized in both ground and excited states. For the excited state optimization, the UV-Vis spectrum was calculated within the Time-Dependent Density Functional Theory (TDDFT) methodology using the same functional as for the optimizations72. The number of excited states included in the TDDFT calculations is 20. We performed TDDFT geometry optimizations in order to find the minimum energy point on the excited state potential energy surface in all cases (vacuum and solvents) using the HOMO-LUMO excitation peak. The SMD solvation model from Truhlar and coworkers was used to model the different studied solvents.All Density Functional Theory (DFT) calculations were carried out with Gaussian 09 (revision D.01) electronic structure package with a 102. An MTT cell proliferation assay was performed as previously reported with noted changes73. Briefly, SH-SY5Y cells were plated in 96-well dishes at a density of 30,000 cells/well in 100 µL of EMEM/F12 supplemented with 10% FBS. After adhering overnight, cells were dosed with various concentrations of AACA probes 1–5, with final concentrations ranging from 0–200 µM (1% DMSO) for 24 hours at 37 °C. After 24 hours of incubation, 20 μL of the MTT reagent (Biotium) was added to each well and the cells were let incubate at 37 °C for 2 additional hours. All solutions were then removed and the formazan crystals were subsequently solubilized with DMSO (100 μL). The cell viability was determined by measuring the absorbance at 570 nm using a Molecular Devices SpectraMax i3 multimode plate reader. All results were expressed as percent reduction of MTT relative to control cells (defined as 100% viability) and the average absorbance value for each treatment was blanked with the absorbance reading of media control blanks. Each concentration was done in at least triplicate.SH-SY5Y cells (ATCC # CRL-2266) were cultured in 1:1 Eagle’s Minimum Essential Medium (EMEM): F12 medium supplemented with 10% FBS and maintained at 37 °C in a humidified atmosphere with 5% CO74.Aggregated Aβ peptide was prepared by dissolving Aβ (1–42) in 1xPBS pH 7.4 to a final concentration of 100 µM. This solution was stirred at 1200 rpm for 3 days at room temperature. The aggregation state was verified by a standard thioflavin T fluorescence assayα-Synuclein aggregates were similarly prepared by dissolving α-synuclein in sterile 1xPBS pH 7.4 to a final concentration of 100 µM. Solutions were incubated at 37 °C for 7 days prior to use. The presence amyloid fibrils was verified by standard thioflavin T fluorescence assay.75. Briefly, monomeric insulin was dissolved in pH 1.6 H2O at 65 °C for 8 days. Aggregates were then dialyzed into pH 7.4 1xPBS for 24 hours at 25 °C and subsequently incubated for 48 hours at 65 °C before storing at 37 °C prior to measurements. The presence of amyloid fibrils was verified by a standard thioflavin T fluorescence assay.Insulin amyloid aggregates were prepared using a modified version of the protocol described by Jiménez and coworkers2 to stain neurofibrillary tangles (NFTs) in human brain samples from AD patients (data not shown).Tau was not included in the analysis due to the inability of ANCA compound 1 was excited at 420 nm, probes 2–5 were excited at 450 nm.A 15 μL of stock (100 μM) aggregated amyloid solutions were added to 285 μL of the AACA probes (in 5% DMSO in nanopure water) to attain a final concentration of 5 μM amyloid aggregate and 4 μM of the probe. Concentrations were chosen assuming a 1:1 probe to fibril binding stoichiometry to avoid oversaturation of the fluorimeter detector. The solutions were transferred to a 300 μL cuvette and the fluorescence was measured at 22.5 °C. The excitation wavelength varied depending on the maximal absorbance of the probe; AACA probe 2O: HFIP). Aliquoted fractions were lyophilized for 2 days, followed by storage at −80 °C until use. HFIP treated lyophilized Aβ was solubilized in DMSO (clear solution) to a final concentration of 1 mM. The solution was then diluted into PBS to 100 μM and centrifuged at 14,000 rpm for 10 minutes at 4 °C. The supernatant (t = 0) was taken and allowed to aggregate at 37 °C. At various time points (t = 0–120 hours) aliquots were removed to measure the fluorescence binding of probe 2 with Aβ and to be frozen down for gel analysis. The emission spectra (excitation at 450 nm) were taken using a final concentration of 4 uM of probe 2 with 5 uM Aβ in 5% DMSO/DI H2O  Gel analysis of Aβ was done under non-reducing SDS-PAGE and visualized with silver staining (Pierce Silver Stain Kit).Aβ(1–42) was initially solubilized in 100% HFIP to 1 mM concentration at RT for 24 hours with shaking. The solution was sonicated and vortexed before it was diluted in cold nanopure water  for the probe-Aβ complexes from the fluorescence studies, we made the following assumptions: (1) All probes are completely in solution and free of any significant competing binding process such as self-aggregation. (2) The concentration of unbound probes can be approximated as close to the total concentration of the probes. (3) The binding sites in the aggregated Aβ peptides are not completely occupied at the concentration of Aβ binding probes used for the fluorescence studies .As previously reported1–5 in 5% DMSO in nanopure water and their fluorescence emission recorded. Samples were excited at 420 nm for probe 1, and 450 nm for probes 2–5. The Kd values were determined by fitting data to a one-site specific Hill-binding model   was mixed with various concentrations of probes SweInd, which bears both the Swedish (K670N/M671L) and the Indiana (V717F) mutations, under the control of the Syrian hamster prion protein promoter. Mice were euthanized between 9–13 months of age and their brains extracted into optical cutting temperature (OCT) fixation media. Frozen brain sections were cut from these frozen blocks.Male and female transgenic mice overexpressing wild type mouse PrP (Tga20) were inoculated with the mouse-adapted prion strain mCWD and were euthanized upon developing terminal signs of prion disease. Transgenic mice (19959) harboring the Aβ plaques express the mutant human amyloid precursor protein APP1–5 were diluted 1:50 in 1X PBS (from stock solutions of 3 mM) to a final concentration of 60 μM, added to brain sections, incubated for 30 min at room temperature, washed three times with 1X PBS, and coverslipped using DAKO fluorescent mounting media.Frozen tissue sections were dried for 1 hr, hydrated with 100%, 95%, and 70% ethanol for 5 min each, and then rinsed with deionized water. Sections were then buffered with phosphate-buffered saline (1X PBS) for 30 min. Probes 1) or 488 nm (probes 2–5) laser on an Olympus FluoView FV1000 spectral deconvolution confocal microscope with 5% power under 10X magnification. The emission spectra of probes 1–5 bound to Aβ, PrPSc, and background were collected in 1 nm increments from 450–650 nm. A minimum of 10 measurements was collected for each compound bound to plaque and non-plaque regions as a background control. The wavelength corresponding to the maximum relative fluorescence intensity was taken as the emission λmax. Real color brain sections were imaged under an Olympus MVX10 Macroview equipped with a MWB2 (Japan) long-pass filter and a 2X MVX (PF) Plan Apochromat lens with a 0.5 numerical aperture. Each sample was illuminated with epifluorescence and imaged with an exposure time of 0.2–0.8 sec, depending on the sample brightness.In order to obtain spectral scans of probes bound to amyloid plaques in neural tissue slices, brain samples were excited with a 405 nm (probe m) of the best-fit linear regression lines for equation (m · x + b) were used to extract the binding pocket permittivity (x) from the observed fluorescence emission λmax (y). Slope values for all probes are tabulated in Supplementary Table The calibration curves based on the reduced Lippert-Mataga solvatochromism (equation ) were usequation  .Supplementary Information


The microstructures were investigated by combinations of conventional and aberration-corrected transmission electron microscopy. Diffraction contrast analysis and high resolution imaging reveal that high density interfacial dislocations were distributed at the interfaces. These dislocations have mixed character with Burgers vectors of a <110> and line directions of <112>. The edge components of the dislocations, with the Burgers vectors parallel to the interface, accommodate the lattice mismatch and are the main contributor to the misfit relaxation of this system. The formation mechanism of these dislocations is proposed and discussed to elucidate the novel mismatch relaxation behavior of <111> oriented perovskite films.Ferroelectric thin films grown on high index substrates show unusual structural and switching dynamics due to their special strain states. Understanding the misfit relaxation behavior is crucial to facilitate the high index thin film growth with improved quality. In this paper, ferroelectric PbTiO In multiferroic materials, Chu et al.3 thin film by using substrate orientations as a critical control parameter. Therefore, high-index substrates could not only play an important role in regulating the microstructures of high-index films, but also are important in controlling and obtaining intriguing properties of perovskite oxide thin films.High-index ABO3 perovskite oxides in general which will be an enormous challenge for the growth of epitaxial high-index ABO3 films3 (or LaAlO3) consist of alternating SrO34−and Ti4+(or LaO33−and Al3+) charged planes stacked along the [111] direction. This electrostatic dipole moment normal to the surface yields a divergent surface potentials when the thickness of the films increases, making an unreconstructed (111) surface quite unstable. Next, the epitaxial thin film growth along these highly polar directions may accelerate the occurrence of complex surface reconstructions that act to compensate for the polar mismatch, which will prevent the first (few) monolayers of the films from coherent growth3 heterostructures was recently reportedUnfortunately, a crucial challenge in the implementation of both the theoretical and experimental proposals for those high-index perovskite heterostructures is the difficulty for preparing thin films with high quality along 111] direction. There are many factors to hinder this process. First, high index surfaces involving polar discontinuities tend to increase surface energies for ABO1 directi3 (PTO) thin films on (111)-oriented substrates have been inadequate, even though these tetragonal ferroelectric films grown on such (111) substrates would exhibit novel physical properties different from (001) and (110)-oriented ones3In addition, owing to the strong coupling between strain and ferroelectricity, misfit strains and defect structures can make an enormous impact on the microstructures and properties of displacement ferroelectrics133 (111) (LAO (111)) substrates by pulsed laser deposition (PLD) technique. Misfit dislocations with Burgers vectors of a <110> and line directions of <112> were observed along the interface, which constitute the major interfacial defects in these films. Moreover, the mechanism of relaxing misfit strain between PTO and LAO were discussed. Particularly, the formation mechanism of these novel misfit dislocations is proposed and discussed as well.In this paper, transmission electron microscopy (TEM) and aberration-corrected scanning transmission electron microscopy (STEM), which could provide resolution to the sub-angstrom level and identify different atom columns directly, were used for investigating interfacial structures and strain/defects in PTO (111) films15rh = 5.63 Å and space group of R-3c at room temperaturep = 3.79 Å. In the following, pseudocubic indices will be used and indicated by the subscript “p.” Above Curie temperature, the PTO crystal shows a simple cubic structure with the space group of Pm-3m and the lattice parameter of a = 3.95 Å. On cooling through the Curie temperature, the symmetry of the PTO crystal decrease to a tetragonal structure which belongs to the P4mm space group with the lattice parameters of a = 3.89 Å, c = 4.14 Åp111 = 2.19 Å, dp110 = dp101 = dp011 = 2.68Å, and dp112 = 1.55 Å. The corresponding interplanar spacings in the tetragonal PTO are d111 = 2.29 Å, d110 = 2.75 Å, d101 = d011 = 2.83 Å, d112 = 1.65 Å, and d121 = d211 = 1.60 Å. It turns out that the interplanar spacings of PTO are comparable to the corresponding interplanar spacings of LAOP. The mismatch f is calculated to be −2.58% for dp110 and d110, −3.17% for dp112 and d121/d211, −6.25% for dp112 and d112, and −5.44% for dp110 and d101/d011, respectively. It is thus determined that the as-grown films are under compressive and suffer an anisotropic in-plane strains coming from low symmetry surfaces of LAO substrate.It is well known that bulk LAO is rhombohedral with the lattice parameters of aConventional TEM imaging was performed on Tecnai G2 F30 TEM for acquiring fundamental information of PTO thin films. TEM investigations indicate that the interfacial structures of the PTO/LAO (111) systems are complicated and very different from low-index interfaces.P. The splitting of the spots indicates that mismatch relaxation between the PTO and LAO lattices occurred. Moreover, we can see some extra weak diffraction spots denoted by red arrows which should be from domains (slanted lines in 1b).Electron diffraction patterns clarify that the as-received PTO/LAO film system possesses well orientation relationships as shown in P, respectively. Those lines might be misfit dislocation networks related to the lattice mismatch between the film and the substrate. If they were dislocation networks, the spacing (S) of the parallel dislocations could be given by:In order to clarify the high density black dots near the interface, conventional TEM diffraction contrast analysis was carried out. It is found that the contrast changes dramatically associated with the g vectors. b is the magnitude of Burgers vectors of misfit dislocationsp110 = dp101 = dp011 = 2.68Å. For the tetragonal PTO, d110 = 2.75Å and d101 = d011 = 2.83Å. Thus the theoretical dislocation spacings are about 10 nm and 5 nm for d110 = 2.75Å and d101 = d011 = 2.83Å, respectively. Experimentally, the line spacings in P. This kind of misfit dislocation configuration is very different from the ones in previous studies on (001) and (110)-oriented thin film systems20202122It is noted that the density of misfit dislocation lines is high because of large misfit strains between PTO and LAOTo accurately determine the Burgers vectors, aberration-corrected HAADF-STEM experiments were performed because of their high spacial resolution and high chemical sensitivity.p showing the interface of the PTO/LAO (111) film. An array of misfit dislocations is found nearly periodically distributed along the interface, and the position of each dislocation is denoted by vertical green arrows. The dislocation are spaced about 8~10 nm apart. For showing the locations of dislocations more clearly, the positive strains along the [1p, respectively. As shown in 21a <100>, the corresponding perfect dislocation are probably a <110> type. To clear this point, it is essential to tilt the sample and measure corresponding projected Burgers vector from another direction. p. In this image, it is noted that the misfit dislocation core looks sharp and no blurring, implying that the dislocation is edge-on and possess [11To display the structural details of misfit dislocations, atomic scale HAADF-STEM images were performed as well. It is well known that the misfit dislocation is an important way to relax strains between film and substrate22Here, 1/2a[1To generalize the misfit strain relaxation behavior in PTO/LAO (111) system, we have further prepared films with different thicknesses. Similar strain relax processes via a <110> misfit dislocations were persistently observed, which suggests the above mechanisms are common for PTO/LAO (111) system.110 and d112 are very different. In addition, d112 and d211 are not equivalent as well. Therefore, the film bears anisotropic in-plane strains when grown along <111>. For perovskite film systems, it is generally believed that a misfit dislocation is generated either from the extension of pre-existing dislocations in the substrate or from the half-loop nucleation and expansion from the film surface by dislocation gliding or climbing and dislocation reaction2122et al.It is well known that if the mismatch is relatively small, films can grow coherently on substrates until a critical thickness is reached. When the film thickness exceeds the critical value, the introduction of misfit dislocations will be energetically preferred over fully coherent growth. For the <111> oriented perovskite films, the in-plane lattice constants dIn summary, we have investigated the misfit strain relaxation behavior of (111)-oriented ferroelectric PTO epitaxial films grown on LAO (111). From the structural characterizations by conventional TEM and aberration-corrected TEM observations, we found that the growth of PTO (111) thin films is island-like Volmer-Weber mode due to the large misfit strains. The main approach for relaxing the misfit strains is the formation of misfit dislocations, which have mixed character with Burger vectors of a <110> and line directions of <112>. Only edge dislocation components relieve the misfit strains between the films and substrates. The screw dislocation components relax the shear strains resulting from ferroelectric domains and anisotropic strains from the substrate surfaces. The results reveal that the anisotropic and inhomogeneous in-plane strains coming from low symmetry substrate surfaces cause an unusual misfit relaxation behavior of the epitaxial PTO films. These findings may provide some insight for understanding the anisotropic strain relaxations in other (111)-oriented perovskite films, which demonstrate novel physical functionalities.−2, a laser repetition rate of 4 Hz and under an oxygen pressure of 20 Pa. After deposition, the films were annealed at 650 °C in an oxygen pressure of 2 × 104 Pa for 60 min, and then cooled down to room temperature at a cooling rate of 5 °C min−1.Epitaxial thin films of PTO were grown on well-oriented LAO (111) substrates by PLD. A Lambda Physik LPX 305i KrF (λ = 248 nm) excimer laser was used. Before deposition, the substrate was heated at 750 °C for 20 min to clean the substrate surface and the laser was focused on a ceramic PTO target for 30 min pre-sputtering to clean the target surface. Throughout the deposition process, the substrate temperature was kept at 650 °C, with a laser energy density of 2 JcmTEM specimens for cross-sectional observations were prepared by conventional method by cutting, gluing, grinding, dimpling, and finally ion-milling. A Gatan 656 Dimple Grinder was used for dimpling. Ar-ion-milling was performed by using a Gatan 695 Precision Ion Polishing System. Plane-view TEM samples were thinned and ion-milled only from the LAO substrate side until the Ar ion beam perforated the samples.A Tecnai G2 F30 TEM working at 300 kV, was used for electron diffraction and diffraction contrast analysis.3TM G2 60–300 microscope fitted with a high-brightness field emission gun and a monochromator, and double Cs correctors from CEOS) operating at 300 kV. Strain fields were deduced by using custom plugins of geometrical phase analysis (GPA) for Gatan DigitalMicrograph27Atomic resolution high-angle annular dark field (HAADF) STEM images in this study were recorded using aberration-corrected TEM  Thin Film System. Sci. Rep. 6, 35172; doi: 10.1038/srep35172 (2016).


Medicago truncatula is regulated by the nitrogen status of leaves through inducing a repeatedly occurring 24-h nodule activity rhythm that reduces per day nitrogen fixation. The hypotheses of the present study were that (1) long-term moderate whole-plant P deficiency in Medicago truncatula induces an according daily rhythm in nitrogenase activity comparable to that induced by nitrate application and (2), the changes in the nodule transcriptome that go along with a strong nitrogenase activity decline during the afternoon would be similar under P deficiency or after nitrate supply. The nodules of plants in a low P treatment developed a rhythmic pattern of activity that resembled the pattern following nitrate application. A comprehensive, RNAseq-based comparative transcriptome profiling of nodules during a repeated part of the rhythm revealed similarities between P deficiency versus nitrate supply. Under both treatments, the formation of nitrogenase was targeted by a reduction in the expression of genes for nodule-specific cysteine-rich peptides (NCR), and possibly also by a disturbance of the inner cell iron allocation. A strong reduction in the expression of leghemoglobin is likely to have restricted the supply of oxygen for respiration.Nitrogen fixation of  Estimates based on detailed measurements of the carbon balance in a greenhouse experiment reveal the energy cost of driving nitrogenase to be approximately equivalent to 25% of the shoot dry matter at harvestSymbiotic nitrogen fixation is a cornerstone of sustainability in worldwide agriculture, but is a demanding process for legume plants, both with regard to assimilate and nutrient needs for nodule formation and also in terms of energy and carbon expenditure for the N2 reduction. The second is the ability to reduce nitrogen fixation of existing nodules quickly and efficiently if any alternative N source becomes available or any environmental cue restricts growth. The principal steps forming the molecular basis for the tight regulation of the nodule number are widely understood346891114The legume plant has two main mechanisms for applying the above basic rules for the symbioses. The first is regulation of the number of nodules and thus the available ‘machinery’ for symbiotic NMedicago truncatula induces an according daily rhythm in nitrogenase activity comparable to that induced by nitrate application and (2), the molecular reason for a strong nitrogenase activity decline during the afternoon – a major part of the daily rhythm – would be similar under P deficiency or after nitrate supply.Based on the striking similarity of the pattern in the 24-h rhythm of nodule activity between nitrate application and other treatments that reduce nitrogenase activity10M. truncatula plants in nutrient solution with limited P supply to produce lower growth but keep the plants alive. The growth system was embedded in a measurement system that allowed continuous, non-invasive monitoring of nitrogenase activity by measuring nodule H2 evolutionTo validate these hypotheses, a procedure was established to grow M. truncatula previously inoculated with Sinorhizobium meliloti (102F51) and with visible active nodules were grown in a quasi flow-through nutrient solution system, where the P concentration was adjusted daily. Using this system, a daily adjustment of the P concentration to 5 μM proved optimal for growth of M. truncatula when depending solely on nitrogen fixation for N supply. A daily measurement of the P concentration before the adjustment to 5 μM showed that the concentration was never depleted below 3 μM P during the experiment (eight weeks of growth), thus allowing the plants a continuous optimal P uptake and N2-fixation. As a treatment with limited P supply (P deficiency), a daily adjustment of the nutrient solution to 1 μM (20% of full P supply) was chosen. The daily P concentration before the adjustment of the solution showed that the plants reduced the concentration to a minimum value below which the plants cannot take up P anymoreLmin value [0.2–0.3 μM]) throughout the experiment and thus suffered a limited P supply. The preliminary experiment had shown that M. truncatula plants under these conditions experienced reduced growth, but maintained a certain level of nitrogen fixation and did not die during the planned eight-week experimental period. Dry matter (DM) formation, nodule number and N and P concentrations in the various organs of the plants are shown in 181920Plants of versus a greenish to brown colour of the nodules. Although we are not aware of any direct measurement that greenish nodules have totally lost their nitrogenase activity, the fact that the green colour results from a decay product of leghemoglobins, which are of vital importance in nodule functioning, makes this highly probable. In any event, when considering the entirety of the collected nodules, the gap between the control and low-P treatment would even have widened since the share of inactive nodules was higher in low-P plants.Nitrogen fixation was measured continuously during one day in the eighth week of growth. While the plants in the control showed a stable nitrogen fixation throughout a 24-h period, the low-P plants had developed the typical daily rhythm. In particular, a strong decline occurred during the time between about noon and 5 pm . Nitrogeversus those from plants grown under limited P supply. The data of the comparison nitrate versus control are published in Cabeza et al.M. truncatula (Mt4.0v1 versus Mt3.5v3 that was previously used). The reason for that approach was to form a basis for comparison of the molecular reasons for the ‘afternoon decline’ induced by long-term P deficiency versus nitrate application. The harvest in both treatments was done at the exact same time, at which in both cases the ‘afternoon decline’ was in full effect  was again found with the new cds annotation. The importance of the gene for efficient nitrogen fixation and for nodule acclimation to altered external oxygen pressure has recently been shownThe main described effects of nitrate on the nodule transcriptome2FC greater than [1], meaning that the abundance of the mRNA was at least doubled or halved. A quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation proved the reliability of the RNAseq data (2 = 0.89). The slope of the regression line was close to one (0.94).Nodules for the RNAseq analysis were harvested during the P deficiency induced ‘afternoon decline’ parallel with the nodules harvested 4 h after nitrate application see . The seqseq data : sheet 52-fixation that clearly showed a 24-h rhythm of nitrogenase activity. The pattern of the rhythm was consistent with that under other treatments2019The low-P treatment resulted in P-deficient plants that could be kept alive during eight weeks of growth in the present study’s experimental system. The plants showed typical growth reactions to limited P supply2120versus (over)-sufficient nitrate application  (2FC numbers showed a close correlation (r2 = 0.89) and the predicted regression line was close to a 1:1 correlation  : sheet 1relation .2FC of significantly and commonly regulated TUs of both treatments were weighted together by the RPKM (Reads Per Kilobase per Million mapped reads) values of the control (RPKMcontrol × log2FC after 4-h nitrate impact × log2FC after eight weeks of low P supply) the above-mentioned Medtr1g084050.1, annotated as ‘nicotianamine synthase-like protein’, was the most strongly affected TU  and dnf7 are impaired in the functioning of the gene Medtr4g035705 and Medtr7g029760 respectively. Both genes encode an NCR-peptide332FC Medtr4g035705.1 −0.8 or −0.59 under nitrate or P deficiency respectively; log2FC Medtr7g029760.1 −1.42 or −0.9 under nitrate or P deficiency respectively). Nevertheless, the expression and also the weighted log2FCs in transcript abundance of both TUs for the two NCR-peptides were only to a medium extent when considering the numerous other affected TUs for NCR-peptides. This fact might support the specific individual importance of the different NCR-peptides. On the other hand, in an earlier study, the individual knockdown or overexpression of five different genes for NCR-peptides resulted in no detectable phenotype, suggesting a certain redundancy among the NCR-peptidesdnf1), which encodes for a core unit of this complex, results in fix− nodulesThere were a total of 203 TUs for NCR-peptides that were downregulated under both treatments from a high level of expression in the nodules. In fact, when the log2 conductance. While nitrate has been shown to lead to lower O2 influx into the nodule and lower partial oxygenation of leghemoglobin2 conductance increases under P deficiency252 buffering is not the primary function of leghemoglobin. Instead the lower expression of leghemoglobins might be a mechanism for lowering nodule and bacteroid respiration and thus ATP production for driving nitrogenase. RNAi interference with a gene for leghemoglobin in Lotus japonicus resulted in a fix− phenotype10162 binding and bufferingA further clear common effect of the treatments was the concerted downregulation of almost all the expressed genes for TUs of leghemoglobin. Eleven of the 16 genes annotated as leghemoglobins in the cds annotation Mt4.0 v1 were expressed and nine of them were strongly downregulated by both treatments in the nodules in the present experiments : sheet 3In conclusion, the data from the present study supported the fact that the downregulation of nitrogenase under conditions of over-sufficient whole-plant N availability (induced by nitrate application or P deficiency) initially occurs through a 24-h rhythmic pattern of nodule activity. This pattern is induced by common molecular mechanisms in the nodule directly involved in nitrogenase formation and respiration.Medicago truncatula (Gaertn.) cv. Jemalong A17 were submerged in H2SO4 (96%) for 5 min for chemical scarification, sterilised with 5% (v/v) sodium hypochlorite for 3 min and rinsed several times with deionised water. The seeds were subsequently kept at 4 °C for 12 h in darkness, submerged in tap water. The next step was a two-to-four day slight shaking of the submerged seeds at 25 °C and continuous light. When the seeds had developed a ca. 20-mm-long primary root, they were transferred to small growth boxes (170 mm × 125 mm × 50 mm) filled with aerated nutrient solution. Each box contained 20 seedlings, which were fixed by small x-shaped cuts in the adhesive tape on the upper side of the boxes.Seeds of −2 s−1. Immediately after being transferred to the growth boxes, the seedlings were inoculated with 1 mL box−1 of a stationary Sinorhizobium meliloti (Sm) (102F51) YEM-culture of an approximate cell density of 109 mL−1. The Sm strain induced good nodulation with the first macroscopic nodules visible after about 7–10 days. This strain does not contain an uptake hydrogenaseThe plants were grown for two weeks in these boxes in a growth chamber with a 16/8-hour light/dark cycle at 25/18 °C respectively. The nutrient solution level in the boxes was maintained by the addition of an appropriate amount of nutrient solution every other day. The light intensity at plant height was approximately 400 μmol m2 and CO2 evolution. The system is described by Fischinger and Schulzeet al.via plant transpiration could be adjusted by the addition of nutrient solution to the container, thereby not interfering with the measurements in the root/nodule compartment. In addition, a pump in the container drove a nutrient solution flow of about 10 mL min−1 into the upper part of each individual glass tube−1 . The nutrient solution contained macronutrients (mM) K2SO4, 0.7; MgSO4, 0.5; CaCl2, 0.8 and micronutrients (μM) H3BO3, 4.0; Na2MoO4, 0.1; ZnSO4, 1.0; MnCl2, 2.0; CoCl2, 0.2; CuCl2, 1.0 and FeNaEDTA , 30. pH was adjusted to 6.4 using KOH and buffered with 0.25 mM 2-(N-morpholino) ethane-sulfonic acid (MES). After transfer to the growth boxes, the nutrient solution was adjusted once to a 0.5 mM NH4+ concentration by the addition of (NH4)2SO4 since low concentrations of ammonium support growth and nodule formation in young M. truncatula plants2PO4 to the nutrient solution to adjust a concentration of 5 μM P for plants in the control and the nitrate experiment. The nutrient solution in the low-P treatment was adjusted daily to 1 μM P.After two weeks, the plants were transferred to glass tubes, allowing the separate measurement of root/nodule HThe nutrient solution was changed every week. During this procedure, the pump in the container was switched off and the backflow from the glass tubes to the container was blocked. Using this method, the ongoing measurements in the root/nodule compartment were not affected. The procedure of replacing the nutrient solution in the container took about ten minutes, after which the nutrient solution turnover system was set back to normal.3 at 180 °C and the P concentration in the digest was measured colorimetrically by the molybdenum-vanadate methodPlants were dried to constant weight at 65 °C before the dry matter was determined. Subsamples of plant dry matter were digested in concentrated HNO2 evolution, including the determination of apparent nitrogenase activity (ANA), total nitrogenase activity (TNA) and the calculation of the electron allocation coefficient (EAC), is described by Fischinger and Schulze2 concentration data in the continuous gas stream were taken every 5 min. TNA was measured on parallel plants at various points in time during the experiment.The system for measuring nodule Hhttp://www.ncbi.nlm.nih.gov/tools/primer-blast). To validate the RNAseq results, RNA from the RNA pools previously used for Illumina sequencing was used. The samples and reference genes were run in triplicate using the Fast SYBR Green Master Mix protocol  on a StepOne™ Real-Time PCR System  following the manufacturer’s recommendations.For the quantitative reverse transcription-polymerase chain reaction (qRT-PCR), gene-specific primers were designed using Primer-Blast , 2 μL reverse primer (2 pmol/μL) and 2 μL template cDNA (100ng). The PCR conditions according to the Fast SYBR Green Master Mix protocol were 20 seconds of pre-denaturation at 95 °C, 45 cycles of 3 seconds at 95 °C and 30 sec at 60 °C, followed by steps for the dissociation curve generation . StepOnePlus software  was used for data collection. Relative transcript levels were obtained using the comparative CT method (ΔΔCT).qRT-PCR was performed in a 20-μL reaction mix containing 10 μL Total RNA was purified from 27 samples [ ×  × ] using the TRIzol protocol . RNA was digested with RNAse-free DNAse  and checked for integrity by capillary gel electrophoresis .Library preparation for RNAseq was performed using the TruSeq RNA Sample Preparation Kit  starting from 500 ng of total RNA. Accurate quantitation of cDNA libraries was performed using the QuantiFluor™ dsDNA System . The size range of the final cDNA libraries was determined by applying the DNA 1000 chip on the Bioanalyzer 2100 from Agilent  (280 bp). The cDNA libraries were amplified and sequenced using the cBot and HiSeq2000 from Illumina .The sequence images were transformed with the Illumina software BaseCaller to bcl-files, which were demultiplexed to fastq files with CASAVA v1.8.2. Quality check of raw data was performed with the FastQC Software , in order to assess the overall and per-base quality of reads. Illumina adapters were removed with the Trimmomatic Softwaret-test. Cluster analysis for the dendrogram .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Our understanding of new genes is often built upon the knowledge of well-characterized genes. One avenue toward revealing such connections involves creation of strains with mutations in two or more defined genes to permit genetic interaction analysis. Strain manipulations can yield unexpected mutations at loci outside the defined targeted genes. In this report, we describe a method for rapid validation of multigene mutants, thus allowing an appraisal of the contribution of the defined targeted genes to the strain’s phenotype. in vivo recombinational assembly strategy that does not require cloning at all. Our results show that CARMA allows genetic rescue of two double-gene mutant strains of the fungal pathogen Candida albicans.The biological function of a gene is often probed through its interactions with other genes. This general approach has been especially useful to build knowledge about poorly understood genes upon the bedrock of well-characterized genes. Genetic interaction analysis requires the construction of strains with mutations in two or more genes. Single-gene mutants of microbial pathogens are generally validated through introduction of a wild-type copy of the affected gene to create a complemented or reconstituted strain, followed by testing for restoration of a wild-type phenotype. This practice, formalized as one of Falkow’s “molecular Koch’s postulates” ensures that the phenotype of the mutant depends upon the known mutation. However, multigene mutants are seldom validated because of the labor required to assemble multiple genomic segments into a vector that can be introduced into the mutant strain. We present here an approach, concatemer assembly for rescue of mutant abilities (CARMA), that circumvents this impediment through an IMPORTANCE Our understanding of new genes is often built upon the knowledge of well-characterized genes. One avenue toward revealing such connections involves creation of strains with mutations in two or more defined genes to permit genetic interaction analysis. Strain manipulations can yield unexpected mutations at loci outside the defined targeted genes. In this report, we describe a method for rapid validation of multigene mutants, thus allowing an appraisal of the contribution of the defined targeted genes to the strain’s phenotype. For many organisms, our understanding of gene function is based upon analysis of engineered mutations at predetermined genomic sites. The manipulations that yield engineered mutations can also yield adventitious mutations elsewhere in the genome. For this reason, when characterizing a defined mutant strain, there is a concern that its phenotype may be due to adventitious mutations, rather than the engineered mutation. For microbial pathogens, mutant strain characterization can be extensive, involving, for example, animal infection models. Moreover, strain phenotypes may appear to be complex because many mechanistic underpinnings of virulence have yet to be discovered. For these organisms, the possible contributions of adventitious mutations to the phenotype are often assessed through analysis of a complemented or reconstituted strain, in which a wild-type copy of the gene that had been altered by engineering is introduced at a distant genomic site (complementation) or at the native locus (reconstitution). The importance of including complemented or reconstituted strains in pathogen phenotypic characterization was emphasized as the third postulate among the cornerstone “molecular Koch’s postulates” coined by Falkow .Saccharomyces cerevisiae, systematic gene interaction analysis has been applied on a large scale to reveal new gene functions and novel connections among seemingly distinct biological processes -Cas9-based methods , has the potential to accelerate multigene mutant validation in C. albicans and other organisms.Here, we present a genetic rescue strategy that relies on recombinational assembly in YFG1 and YFG2 genes to assay phenotypic rescue of a yfg1Δ/Δ yfg2Δ/Δ double mutant. We include the marker M1 in order to select for transformation and integration of a concatemer of the three segments. Each segment is synthesized through a PCR templated by the genome or an existing plasmid, thus eliminating any need for custom cloning. The segments have homology at their ends to one another or to the genome  segment includes one of the coding regions, e mutant . It alsoYFG2, that corresponds to a deletion in the yfg1Δ/Δ yfg2Δ/Δ double mutant  segment includes the second coding region, e mutant . This seM1, along with all necessary 5′ and 3′ flanking sequences for expression (YFG2 segment (D-E region) at one end and to the genome neighboring the yfg1Δ allele (F region) at the other end. The 5′-to-3′ orientation of each gene  is arbitrary; the diagram illustrates the actual relative orientations of the genes in the experiments we present below.The third (rightmost) segment includes a marker for selection, pression . It has M1 on its own. However, the YFG1 and YFG2 segments may serve as adaptors to enable homologous recombination events to insert a YFG1:YFG2:M1 concatenated assembly in place of the yfg1Δ allele  that targets the 300-fold . Approxiume6Δ/Δ brg1Δ/Δ double mutant strain. UME6 and BRG1 encode transcription factors that promote C. albicans filamentation and biofilm formation (–UME6 segment (YFG1), a BRG1 segment (YFG2), and a C. dubliniensisHIS1 segment (M1). Integration of the concatenated cassettes was targeted to the ume6Δ::r1 locus. The ume6Δ::r1 locus has a deletion of the UME6 coding region; it contains the UME6 upstream and downstream regions flanking a 360-bp segment (designated r1) of vector pRS424  e3 plus 4  and C) e5 plus 6  and D) e7 plus 8  and E) eume6Δ::r1 alleles .  plus 10 ). These UME6:BRG1:HIS1 concatenation product and was presumably homozygous due to the absence of a ume6Δ::r1 allele. We refer to this strain as the validation strain. Three assays were used to assess phenotypic rescue: filamentation, biofilm formation, and expression of UME6- and BRG1-responsive genes  each single mutant has a filamentation defect and (ii) the UME6:BRG1 concatemer restores filamentation when introduced into the ume6Δ/Δ brg1Δ/Δ background.Filamentation was assayed by microscopic observation after growth in yeast extract-peptone-dextrose (YPD) plus serum at 37°C . The wilume6Δ/Δ brg1Δ/Δ mutant was severely defective in biofilm formation: few cells adhered to the silicone surface, and no hyphae were apparent  each single mutant has a biofilm defect, and (ii) the UME6:BRG1 concatemer restores biofilm when introduced into the ume6Δ/Δ brg1Δ/Δ background. However, the UME6:BRG1 concatemer enables only partial rescue of the biofilm defect, because the validation strain does not produce as extensive a biofilm as the wild-type strain does.Biofilm formation was assayed by confocal microscopy after growth on silicone squares in YPD alone at 37°C for 24 h . The wilapparent  and B. Tbiofilms . The UMEjections  and B. TP < 0.05) than the ume6Δ/Δ brg1Δ/Δ double mutant for 18 genes in addition to the deleted genes UME6 and BRG1 . The recipient strains in the present study were all homozygous for identical mutant alleles at the target integration locus. Therefore, for existing yfg1Δ::ARG4/yfg1Δ::URA3 strains, it may be necessary to use two sgRNA genes in order to direct integration to both alleles.Note that, in all experiments reported here, the target integration loci are deletion alleles with fairly small relics of the initial marker; they are A few features of CARMA seem especially useful. First, all of the DNA molecules in the transformation reaction are PCR products, including the DNA segments destined to be assembled into the integrated concatemer, and the genes specifying Cas9 and the sgRNA, which are the original constructs used by Vyas et al.  used witC. albicans strains were all archived in 15% glycerol stocks stored at −80°C. Strains were grown on solid yeast extract-peptone-dextrose (YPD) medium for 2 days at 30°C and then in liquid YPD medium overnight at 30°C with shaking prior to all assays and transformations. Transformations were performed using the lithium acetate transformation method supplemented with transient CRISPR-Cas9 system components  of 0.2 in 250-ml Erlenmeyer flasks. Cultures were then allowed to grow at 4 h with vigorous shaking (200 rpm) at 37°C, then collected by vacuum filtration, and stored at −80°C until ready for extraction. RNA was extracted from frozen cell cultures using the Qiagen RNeasy minikit  with modifications as described previously (Overnight cultures of the relevant strains were inoculated into 25 ml of YPD to a final optical density at 600 nm (ODeviously .Nanostring analysis of RNA samples was performed using previously described methods . The NanBiofilms were grown according to previously described protocols with the following modifications . OvernigThe filamentation assay was performed using strains inoculated into 5 ml YPD in glass test tubes and grown for 4 h at 37°C in a roller drum at maximum speed. Following growth, the cells were fixed and stained with Calcofluor white. An aliquot of cells was then imaged using conventional fluorescence microscopy on a Zeiss Axio observer Z.1 fluorescence microscope and a 20× 0.8-NA objective.Colony counts were collected using OpenCFU software . MicroscNanostring transcription profiling data may be found in 10.1128/mSphere.00169-18.3TABLE S3 TABLE S3, XLSX file, 0.2 MB.Nanostring measurements of RNA levels. Raw data, normalized probe counts, and gene expression ratios are given on three different tabs. Download Copyright © 2018 Huang et al.2018Huang et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the