Optogenetic effector and sensor function was demonstrated in numerous tissues. Arterial/arteriolar tone was modulated by optical activation of the second messengers InsP3 (optoα1AR) and cAMP (optoß2AR), or Ca2+-permeant membrane channels (CatCh2) in smooth muscle (Acta2) and endothelium (Cdh5). Cardiac activation was separately controlled through activation of nodal/conducting cells or cardiac myocytes. We demonstrate combined effector and sensor function in biallelic mouse crosses: optical cardiac pacing and simultaneous cardiomyocyte Ca2+ imaging in Hcn4BAC-CatCh2/Myh6-GCaMP8 crosses. These experiments highlight the potential of these mice to explore cellular signaling in vivo, in complex tissue networks.Optogenetic effectors and sensors provide a novel real-time window into complex physiological processes, enabling determination of molecular signaling processes within functioning cellular networks. However, the combination of these optical tools in mice is made practical by construction of genetic lines that are optically compatible and genetically tractable. We present a new toolbox of 21 mouse lines with lineage-specific expression of optogenetic effectors and sensors for direct biallelic combination, avoiding the multiallelic requirement of Cre recombinase -mediated DNA recombination, focusing on models relevant for cardiovascular biology. Optogenetic effectors (11 lines) or Ca Optogenetic effectors and sensors enable the interrogation of complex biological signaling networks at the molecular level in vivo . The powCHROMus  mice, 21 lines of optogenetic sensor and effector mice, in which the expression of the transgene is lineage specified  element and bacterial LacZ ORF for simple screening. The fluorescent GECI proteins, GCaMP and RCaMP, in reporter lines were derived from circularly permutated EGFP and RFP constructs for in vivo, real-time reporting of cellular Ca2+ signaling, as shown in We report the generation of pecified  and demopecified , optoß2Apecified , and Catpecified  for ligh2+ signaling in response to various physiological stimuli. However, the system provides the novel opportunity to create novel effector/reporter mouse combinations to study the complex network of physiological signals and responses, as illustrated in CHROMus mice can be used directly as individual optogenetic effector or fluorescent reporter lines to study real-time response to light activation of the target tissue or to study cellular or subcellular CaHcn4BAC-CatCh2_IRES_lacZ mouse line, where the expression of the optoeffector CatCh2 and a marker protein (LacZ) are under transcriptional control of the Hcn4 locus in a BAC  underlies the pacemaker current in the SA node, controlling heart rate and heart rhythm . For ligin a BAC , thus di network . LacZ-ex network  and alon network . Express network .To confirm optical control of heart rhythm, we stimulated the SA node area with 473 nm light in vivo while recording the ECG. As shown in Hcn4BAC-CatCh2_IRES_LacZ and Myh6-GCaMP8 mice. Pacing by laser stimulation resulted in coupling between optical stimulation and GCaMP8 fluorescence, as shown in Hcn4BAC-CatCh2 mice could also be mated to Acta2BAC-GCaMP_GR  and Cdh5BAC-GCaMP8 lines  to study smooth muscle or endothelial cell responses to the cardiac pacing. GCaMP_GR (for green/red) is a ratiometric fusion protein consisting of an enhanced GCaMP sequence fused to mCherry  mediates smooth muscle contraction and relaxation, respectively, we examined constriction or dilation of isolated cerebral and mesenteric arteries in response to optical activation of the chimeric Optoα1AR and Optoß2AR proteins. Brief exposure of arteries isolated from Acta2BAC-Optoα1AR_IRES_lacZ mice to 405 nm light resulted in vasoconstriction  was activated at 10 s intervals by the application of a 500 ms step depolarization from the holding potential of –70 mV to +60 mV. Activation of the Optoß2AR protein by 488 nm light increased the amplitude of IK 10 s after the exposure, reaching a higher, relatively stable plateau approximately 80 s after photoactivation  after the laser was turned off. Notably, a second 488 nm light pulse to the same cell evoked an ICat that lacked the high-amplitude transient component of the first light pulse but had the amplitude and kinetics of the steady-state ICat in the first pulse. Similar observations were made in isolated mesenteric myocytes . As shown in Cdh5 gene locus. As with smooth muscle, Optoα1AR, Optoß2AR, and CatCh2 cDNAs and LacZ ORF were targeted to the Cdh5 initiation codon, with an IRES and the LacZ sequence downstream of the optoeffectors. X-gal staining revealed wide expression in endothelial cells of the heart  is expressed in specialized cells of the cardiac conduction system and in arterial endothelial cells, and expression in embryonic myocytes is critical for heart function prior to development of a mature conduction system  gene locus was similarly used to target glial cells. Aldh1l1BAC-Optoα1AR_IRES_lacZ mouse express in cerebellar  and The Jackson Laboratory.We have generated 21 lines of optogenetic sensor and effector mice, in which the expression of the optogene is lineage specified in cardiovascular and related cell lineages , and senCre recombinase driver mice to confer tissue-specific expression of fluorescent markers or optogenetic sensors downstream of LoxP-STOP-LoxP sequences  Cre-driver and LoxP flanked responder lines result from targeted integration within a known locus. Thus a drawback of CHROMus lines is the unknown site of integration, which could result in integration site-dependent effects on the transcription of other genes, or impact the transcriptional fidelity of the inserted optogenetic cDNA due to positional effects. While we have verified the insertion of BAC or cDNA sequences in these lines, and confirmed the viable breeding of the reported lines, these effects cannot be completely known and thus the lines should be used where they provide functional value, but may not be useful in all circumstances. One example is the  protein . FinallyHcn4BAC-CatCh2_IRES_LacZ) and myocyte sensor (Myh6-GCaMP8 strains) mice, we demonstrate the feasibility of combinatorial optogenetics and report the ability to activate and detect heart signaling  , or prev2; 250 µM potassium ferricyanide; 250 µM potassium ferrocyanide) at 37°C for either 4 hr or overnight. For whole-embryo staining, the embryos were fixed in 4% PFA (pH 7.0) for 30 min on ice and washed three times in detergent wash buffer  for 20 min. Whole tissues were fixed in 4% PFA for 2 hr on ice, washed three times in 1× PBS for 10 min and stained in X-gal. For frozen sections, the fixed tissues were dehydrated in 30% sucrose (1× PBS), embedded in OCT, and sectioned at 8 µm. The sections were rinsed in 1× PBS for 5 min (3×) and stained in X-gal.LacZ expression from the optogenetic transgene constructs were screened by X-gal staining . The rinsed heart was cannulated and perfused with warm Tyrode’s buffer . GCaMP8 fluorescence was captured using an Andor-iXon CCD camera mounted on an Olympus MVX10 fluorescent microscope .Mice were given 100 U of heparin IV prior to euthanasia by pentobarbital . The heart was quickly excised with aorta and vena cava vessels attached and rinsed in ice-cold Tyrode’s buffer , and maintained on 1.5% isoflurane. 5% glucose (0.1 mL/10 g) and atropine sulfate (5 µg/100 g body weight) were injected subcutaneously and body temperature was maintained at 37.5°C with a heating pad. The heart was surgically exposed by dissection of the sternum and diaphragm, and ribs retracted for optical access to the thoracic cavity. For optical stimulation of the SA node, mice were positioned on their back on a stereotaxic stage, the pericardial sac was removed, and the thymus gland repositioned from covering the right atrium. Illumination of the node was performed using a 105 µm core fiber (Thorlabs) coupled to a 473 nm continuous wave, diode-pumped solid-state laser system . The tip of the laser fiber was inserted through 500 µm metal tubing and clamped within a four-way axis micromanipulator that allowed positioning of the fiber tip on the anterior junction of the right atrium and superior vena cava. For stimulation of the left ventricle free wall, mice were positioned on their right side and a left thoracotomy was performed between ribs 7 and 8 to gain access to the heart. The tip of the optical fiber was coupled to a collimating lens mount that was positioned to direct the beam at the left ventricle. A customized titanium probe affixed to the stereotaxic stage was adhered (Vetbond) to the left ventricle free wall to minimize movement associated with respiratory and cardiac contractions. Electrocardiogram (ECG) electrodes were attached to 21-gauge needles inserted subcutaneously through the front and contralateral hind limb and recorded with an isolated differential amplifier . ECG and lung pressure traces were continuously monitored through an oscilloscope. Laser pulses were controlled through a TTL signal delivered by a function generator (Agilent 33210A) and signals digitized and recorded on a computer using MATLAB.Mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection, intubated with a 22-gauge cannula, ventilated  (when necessary) resample non-continuous recordings, (2) lowpass filter ECGs with a passband frequency of 100 Hz, (3) invert, scale, and shift ECGs to have a median value of zero and a maximum amplitude of 1, and (4) binarize laser pulse recordings. ECG R-wave peaks were detected automatically using MATLAB’s The monochrome fluorescent digital images obtained from Leica DMI6000B/Q-imaging or Olympus MVX-10/Andor-iXon CCD cameras were colorized using the Lookup Table command in ImageJ.The raw data for vessel width from MATLAB analysis were transformed using the running average algorithm in SigmaPlot 11 and the transformed data were copied to Excel spreadsheet. The net change in vessel width was calculated by finding the minimum width in the first 10 s after laser activation using the MIN function in Microsoft Excel and subtracting this value from SigmaPlot transformed data.Cdh5BAC-Optoα1AR and C57BL/6J animals were determined and compared using the Holm–Sedak method (SigmaPlot v 11).The % change in vessel width was calculated by first determining the maximum width during laser activation using the MAX function in Microsoft Excel and using the formula ((MAX – MIN)/MIN) * 100. The % change in vessel width from five femoral vessels from 2+-PSS before being recorded.All current recordings were acquired at room temperature using an Axopatch200B amplifier and a Digidata1440 digitizer . Borosilicate patch pipettes were pulled and polished to resistance of 3–6 MΩ for all experiments using a micropipette puller . Freshly dissociated arterial myocytes were kept in ice-cold Mg+ (IK) and light-activated CatCh2 (ICat) currents were recorded using conventional whole-cell configuration of the voltage-clamp technique. Currents were recorded at a frequency of 50 kHz and low-pass filtered at 2 kHz. IK and ICat were recorded while cells were exposed to an external solution composed of 130 mM NaCl, 5 mM KCl, 3 mM MgCl2, 10 mM glucose, and 10 mM HEPES adjusted to 7.4 using NaOH. The pipette solution was composed of 87 mM K-aspartate, 20 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM MgATP, 10 mM EGTA, and 10 mM HEPES adjusted to 7.2 by KOH. A liquid junction potential of 12.7 mV was corrected offline. IK were activated by a series of 500 ms test pulses increasing from –70 mV to +60 mV. ICat was recorded while cells were held at –70 mV before and after exposure to 488 nm light.Voltage-gated K public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Acceptance summary:The manuscript by Lee and co-workers describes the development of 21 unique transgenic mouse lines that express optogenetic sensors and effectors in a cell lineage-specific fashion. The knock-in approach allows the sensors and effectors to be rapidly combined or moved to different backgrounds, such as genetic disease models. Such manipulations are often impractical when using a Cre-based system. This constitutes a vital advantage for many studies. The new mice described here will be very powerful tools to study physiology and alteration in disease models.Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Didier Stainier as the Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Scott Earley (Reviewer #1).Thank you for submitting your article "Genetically Engineered Strains for Combinatorial Cardiovascular Optobiology" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:1) The manuscript would benefit from a transparent discussion of potential problems/limitations associated with the mice described here. For example, is there any evidence for off-target or incomplete expression of sensors or effectors? Are there any issues with the breeding of any of these lines?2) In at least one case, the description on the Jackson Laboratory website did not exactly match the description in the Major Resource Table. Jackson stock number 025406 links to a GCaMP3- mCherry mouse, but this is identified as GCaMP5-mCherry in the Table. Please carefully verify this vital information to avoid confusion.3) As provided for experiments using endothelial optogenetic mice, controls using similar laser direction and parameters should be included for each experimental test of the optogenetic activators.4) A major advantage of using mice of the types described is the ease in which disease models, infection, additional genetic manipulations, environmental factors, aging, etc, can be superimposed upon the mice carrying the optogenetic activators and sensors. This point is mentioned only briefly in the discussion. The manuscript would be strengthened if an example of such an application could be provided in the manuscript. It would be nice to see a graded effect of optogenetic stimulation to provide more evidence of the utility of the approach.5) The probes and effectors are constitutively expressed throughout development. This is different from an inducible LoxP-Stop-LoxP approach. While clearly all the animals are viable, some discussion would be helpful that addresses how expression of a probe (for example a Ca indicator) might potentially alter function. Have the Authors assessed that this is not an issue experimentally?Reviewer #1 (Recommendations for the authors):1. The manuscript would benefit from a transparent discussion of potential problems/limitations associated with the mice described here. For example, is there any evidence for off-target or incomplete expression of sensors or effectors? Are there any issues with the breeding of any of these lines?Anecdotally, the Acta2-GCaMP5-mCherry mice failed to breed in my laboratory, and I've heard from at least one other investigator who had the same issue. I have discussed this with some of the authors of the current study. Have any of the authors of the study encountered such problems with any of the other lines? It would be helpful to future users to disclose these issues and provide practical guidance.2. In at least one case, the description on the Jackson Laboratory website did not exactly match the description in the Major Resource Table. Jackson stock number 025406 links to a GCaMP3- mCherry mouse, but this is identified as GCaMP5-mCherry in the Table. I didn't check them all. I suggest that the authors carefully verify this vital information to avoid confusion. Also, from the Jackson laboratory website, most, if not all, of these strains are cryopreserved. Recovery takes several months and is expensive. It might be helpful to potential users to disclose this limitation.Reviewer #2 (Recommendations for the authors):The paper is well-written and thought provoking. The study is essentially a methods papers with some, if not all, of the mice described having been used in previous investigation-based studies.As provided for experiments using endothelial optogenetic mice, controls using similar laser direction and parameters should be given for each experimental test of the optogenetic activators.While the breeding tactics to generate experimentally useful chimeras are clear, it would also be helpful to compare relative expression/signal strengths for Cre recombinase driver mice vs. the monoallelic mice promoted in the present study. Comments about breeding success/difficulties would also be useful for those planning experiments using combinations of the mice from CHROMus.A major advantage of using mice of the types described is the ease in which disease models, infection, additional genetic manipulations, environmental factors, aging, etc, can be superimposed upon the mice carrying the optogenetic activators and sensors. This point is mentioned only briefly in the discussion, but frankly it is of such importance for future investigation that an example of such an application should be provided in the manuscript.Reviewer #3 (Recommendations for the authors):I have a few suggestions:While the potential for this approach is vast and the data presented validates the approach, no new science or optogenetic/probes are really presented in this paper.It would be nice to see a graded effect of optogenetic stimulation to provide more evidence of the utility of the approach.The probes and effectors are constitutively expressed throughout development. This is different from an inducible LoxP-Stop-LoxP approach. While clearly all the animals are viable, some discussion would be helpful that addresses how expression of a probe (for example a Ca indicator) might potentially alter function. Have the Authors assessed that this is not an issue experimentally? Essential revisions:1) The manuscript would benefit from a transparent discussion of potential problems/limitations associated with the mice described here. For example, is there any evidence for off-target or incomplete expression of sensors or effectors? Are there any issues with the breeding of any of these lines?BAC- CatCh line) is consistent with one report of HCN4 protein expression, and briefly address factors involved in transgene transcriptional control.We have included a new paragraph in the Discussion section addressing this issue (p. 29-30). Briefly, lines were screened for off-target expression and only those whose expression matched the previously reported expression pattern of the underlying gene locus were maintained. We did not observe any instances of off-site expression in the mouse lines that we report. However, while the expected general pattern of expression was matched in these lines, we do not assert absolute transcriptional fidelity. We now cite 3 examples of incomplete expression in the Discussion, although one of these (the HCN42) In at least one case, the description on the Jackson Laboratory website did not exactly match the description in the Major Resource Table. Jackson stock number 025406 links to a GCaMP3- mCherry mouse, but this is identified as GCaMP5-mCherry in the Table. Please carefully verify this vital information to avoid confusion.We thank the reviewer. The Major Resources Table has been revised to include the Jackson Laboratory strain names (as assigned by Mouse Genome Informatics – MGI) in the last column for all strains.3) As provided for experiments using endothelial optogenetic mice, controls using similar laser direction and parameters should be included for each experimental test of the optogenetic activators.We have now included additional controls for optogenetic stimulation experiments .4) A major advantage of using mice of the types described is the ease in which disease models, infection, additional genetic manipulations, environmental factors, aging, etc, can be superimposed upon the mice carrying the optogenetic activators and sensors. This point is mentioned only briefly in the discussion. The manuscript would be strengthened if an example of such an application could be provided in the manuscript. It would be nice to see a graded effect of optogenetic stimulation to provide more evidence of the utility of the approach.We have expanded on this point in the new Discussion section and discussed an example of crossing CHROMus mice with disease models, although such experiments would be the subject of a separate, complete analysis. We also include a graded response to optogenetic stimulation .5) The probes and effectors are constitutively expressed throughout development. This is different from an inducible LoxP-Stop-LoxP approach. While clearly all the animals are viable, some discussion would be helpful that addresses how expression of a probe (for example a Ca indicator) might potentially alter function. Have the Authors assessed that this is not an issue experimentally?We have included this point in the new Discussion section (p. 29-30).Reviewer #1 (Recommendations for the authors):1. The manuscript would benefit from a transparent discussion of potential problems/limitations associated with the mice described here. For example, is there any evidence for off-target or incomplete expression of sensors or effectors? Are there any issues with the breeding of any of these lines?Anecdotally, the Acta2-GCaMP5-mCherry mice failed to breed in my laboratory, and I've heard from at least one other investigator who had the same issue. I have discussed this with some of the authors of the current study. Have any of the authors of the study encountered such problems with any of the other lines? It would be helpful to future users to disclose these issues and provide practical guidance.We are aware of the Acta2-GCaMP5-mCherry (Acta2-GCaMP_GR) breeding problem and include information in the revised discussion.2. In at least one case, the description on the Jackson Laboratory website did not exactly match the description in the Major Resource Table. Jackson stock number 025406 links to a GCaMP3- mCherry mouse, but this is identified as GCaMP5-mCherry in the Table. I didn't check them all. I suggest that the authors carefully verify this vital information to avoid confusion. Also, from the Jackson laboratory website, most, if not all, of these strains are cryopreserved. Recovery takes several months and is expensive. It might be helpful to potential users to disclose this limitation.BAC-GCaMP-mCherry line is Acta2BAC-GCaMP_GR and we have corrected this and the entry for Cx40BAC-GCaMP_GR in Table 1 and Major Resources Table – thank you. The Major Resources Table has been revised to include the Jackson Laboratory strain names (as assigned by Mouse Genome Informatics – MGI) in the last column for all strains. We have now provided the reference for the GCaMP_GR reporter .Thank you. The correct name of Acta2Reviewer #2 (Recommendations for the authors):The paper is well-written and thought provoking. The study is essentially a methods papers with some, if not all, of the mice described having been used in previous investigation-based studies.As provided for experiments using endothelial optogenetic mice, controls using similar laser direction and parameters should be given for each experimental test of the optogenetic activators.While the breeding tactics to generate experimentally useful chimeras are clear, it would also be helpful to compare relative expression/signal strengths for Cre recombinase driver mice vs. the monoallelic mice promoted in the present study. Comments about breeding success/difficulties would also be useful for those planning experiments using combinations of the mice from CHROMus.We have expanded on this point in the discussion.A major advantage of using mice of the types described is the ease in which disease models, infection, additional genetic manipulations, environmental factors, aging, etc, can be superimposed upon the mice carrying the optogenetic activators and sensors. This point is mentioned only briefly in the discussion, but frankly it is of such importance for future investigation that an example of such an application should be provided in the manuscript.We appreciate this suggestion, but we feel that such a study, if done rigorously, should be the subject of a separate, focused analysis, yielding to specific disease insights, rather than an add on to this “toolbox” manuscript.Reviewer #3 (Recommendations for the authors):I have a few suggestions:While the potential for this approach is vast and the data presented validates the approach, no new science or optogenetic/probes are really presented in this paper.It would be nice to see a graded effect of optogenetic stimulation to provide more evidence of the utility of the approach.Thank you. This is now provided in Figure 7C.The probes and effectors are constitutively expressed throughout development. This is different from an inducible LoxP-Stop-LoxP approach. While clearly all the animals are viable, some discussion would be helpful that addresses how expression of a probe (for example a Ca indicator) might potentially alter function. Have the Authors assessed that this is not an issue experimentally?We now provide an expanded discussion of this point.


This is a Phase II randomized controlled trial conducted with the aim of investigating whether the use of Electrochemotherapy after neoadjuvant therapy (ECT) and before surgery in patients with major clinical response allows for a more conservative surgical approach in patients with Locally Advanced Rectal Cancer (LARC) in comparison with the control group that will not receive ECT. The treatment response, in both the control arm and in the treatment arm, will be assessed using the histopathological tumor regression grade on tissue specimens after local excision.Background: Currently, 45–55% of rectal cancer patients receive preoperative chemo- radio-therapy for Locally Advanced Rectal Cancer (LARC). The idea of our study is to use Electrochemotherapy (ECT) before surgery, in patients with major clinical response after neoadjuvant therapy, to allow for a more conservative surgical approach. Objective: To evaluate the increase of the complete response rate after neoadjuvant treatment in LARC and to spare organ function due to total mesorectal excision (TME). Patients and Methods: This is a Phase II randomized controlled trial enrolling 70 patients that will be developed in two stages. In the first step, 28 patients will be enrolled: 14 of these will receive ECT for four weeks after neo-adjuvant treatment and then local excision (treatment group) and 14 patients will receive neo-adjuvant treatment and then local excision (control group). If an increase of response rate is observed in the first stage, and/or feasibility/safety is demonstrated, the second stage of the trial will be performed, enrolling an additional 42 patients. The treatment response. in both the control arm and the treatment arm, will be assessed using the histopathological tumor regression grade on tissue specimens after local excision. Electroporation (EP) is a technique that is able to increase the permeability of the cell membrane by applying a short and intense electrical field that allows molecules that are not, or barely, permeable, to enter into the cell. Electrochemotherapy (ECT) is the combination of a cytotoxic drug and EP.After the first electrochemotherapy clinical trials ,2,3,4, tRecently, the feasibility and benefits of ECT have also been observed and documented in deep solid tumors, such as liver metastasis, primitive liver tumors (cholangiocarcinoma and hepatocellular carcinoma) and pancreatic tumors, both in preclinical and clinical studies ,24,25,26Endoluminal allows the endoscopic treatment of colorectal cancer and gastric and esophageal tumors. This device uses a vacuum to capture tumor tissue while an electroporation pulse is applied. Recently, a phase I clinical trial  on endoshttps://clinicaltrials.gov/ct2/show/NCT03040180, accessed on 10 April 2021). The patients are being treated with bleomycin i.v. according to the ESOPE (European Standard Operating Procedures of Electrochemotherapy) protocol and, in preliminary results, no adverse events or complications have been registered.A phase II clinical trial in non-surgical patients with colorectal cancer is currently ongoing  and 14 patients will receive neo-adjuvant treatment and then local excision (control group). Electrochemotherapy will be performed with the CLINIPORATOR™ and an intravenous dose of Bleomycin. If an increase of response rate is observed in the first stage and/or feasibility/safety is demonstrated, the second stage of the trial will be performed. In the second step, an additional 42 patients will be enrolled and treated. Trial design flow-chart is showed in 1. To assess electrochemotherapy feasibility/safety.2. To evaluate the complete response rate increase with the aim of organ sparing.3. Histopathological analysis evaluation will be based on the tumor regression grade (TRG) by Mandard et al. , performTo evaluate the local recurrence rate, pain control, quality of life and electrochemotherapy toxicity.Eligible patients will be randomly assigned (1:1) to the treatment group or the control group. The treatment group assignment will be assigned blindly and sequentially in the order in which eligible patients are enrolled in the study. Patients eligible for inclusion in the protocol see  will be 2 twice daily, five days a week, for five weeks.Both the Control group and the Treatment group will receive neoadjuvant treatment, either long course radiotherapy with concomitant chemotherapy or short course radiotherapy, before local excision (LE). Long course radiotherapy consists of 45–50 Gy (in fractions of 1.8–2 Gy), with concurrent 5-fluorouracil. Standard fractions of 1.8 Gy/day to the reference point will be given five times a week up to a total dose of 50.4 Gy in 28 fractions . Each suShort course radiotherapy consists of a dose of 25 Gy in five fractions over one week .External radiation therapy will be performed using a 3-field  or IMRT (Intensity Modulated Radiation Therapy) technique.Patients undergoing neoadjuvant treatment for LARC with a major clinical response at restaging will undergo a local excision 16 (±1) weeks after the end of neoadjuvant therapy and histopathological examination with TRG evaluation. Local excision can be performed either with the conventional technique (TAE), or the Transanal endoscopic microsurgery (TEM) or other techniques (TAMIS), provided that the following principles are respected: free macroscopic margin of at least 0.5 cm, full thickness excision, fixation of the operative piece on support to facilitate the histological interpretation of the pathologist.After histopathological examination, based on pathological findings, Total Mesorectal Excision (TME) can then be planned for the patients if, as expected from clinical practice, ypT ≥ 2, there is the presence of positive margins, TRG > 2 and the cancer is poorly differentiated (G3).In the treatment arm, patients undergoing neoadjuvant treatment for LARC with major clinical response at restaging will undergo Electrochemotherapy (ECT) 12 (±1) weeks after the end of neoadjuvant therapy. After four weeks (±5 days) of ECT, the patients will undergo a surgical treatment with local excision and histopathological examination with TRG evaluation.After histopathological examination, based on pathological findings, TME can be planned for the patients.2) and after 8 min, an electric pulse delivered to the lesion will be performed with the CLINIPORATOR™  with the insertion of appropriate CE certificate electrodes for endoluminal treatment [Patients will receive Bleomycin intravenously  and will be recorded. Any clinical or biological abnormalities not present at the trial start will be considered intercurrent events.The visits scheme is summarized in Quality of life (QoL), as measured by the EQ5d questionnaire, will be completed at Visits 1, 2, 3, 5, 6, 7, 8, 9, 10, 11 and 12 and will be considered optional from visit 13 to 18. The EQ-5D was developed in Europe and it is a descriptive system of preference-based Health Related Quality Life . The EQ-All patients will undergo the same enrollment/baseline visit (Visit 1) procedures. Clinical examination will include the measurement of weight, height and vital signs . Patients must sign the informed consent form. The Screening/Enrollment Visit (Visit 1) procedures must be performed not more than 12 (±1) weeks after the end of neoadjuvant therapy. Patients who fulfill all inclusion criteria will be enrolled. Patients who are Eastern Cooperative Oncology Group ECOG (ECOG) Performance Status grade 0, 1 or 2 are eligible for study participation .Study procedures at Visit 1 consist of the following: screening against inclusion/exclusion; randomization to ECT or standard treatment; and Complete Blood Count (CBC) and coagulation profiles evaluation. An absolute neutrophil count below 1000/mL, a platelet count below 70,000/mL, and/or an international normalized ratio (INR) above 1.5 will be considered exclusion criteria. The patients will be subjected to rectal exploration; rectoscopy; routine hematochemistry and carcinoembryonic antigen (CEA), Magnetic Resonance (MR) with contrast medium (cm) and Computed Tomography (CT) with contrast medium. Medical and Oncology history, including any previous treatments, concomitant treatments, medications, and events will be registered. Pain will be evaluated according to the Visual Analog Scale (VAS) scale and Quality of Life will be monitored according to the EQ5d questionnaire.All patients will undergo a pre-anesthetic visit and will be excluded from study participation if not eligible for general anesthesia or monitored anesthesia care (conscious sedation).The major clinical response after neoadjuvant treatment will be evaluated 12 weeks from the end of radiotherapy with the following tests: laboratory exams, CEA dosage, CT of chest and abdomen with contrast medium, MR pelvis with contrast medium and rigid rectoscopy. The major clinical response is classified as the absence of palpable mass to the digital exploration of the rectum and pathological lymph nodes  to the pelvic MRI, and the absence of deep or superficial ulcers > 2 cm of diameter, at rectoscopy.The patients with confirmed eligibility will be treated with ECT. The type of anesthesia will be determined by the anesthesiologist. The patients will undergo clinical evaluation, pain assessment by VAS score and blood samples and CEA dosage. The use of concomitant treatment or drug for pain control will be registered.At day 30 ± 3 (Visit 3), all patients randomized in the control or treatment group will be subjected to LE according to established clinical and institutional practices. The patients will undergo the same procedures as above and according to timing reported in The patients will undergo histological examination; surgical specimens containing the tumor will be evaluated and scored according to TRG, as proposed by Mandard et al. , by an eAfter clinical evaluation, the patients will be submitted to rectoscopy, colonoscopy, pain assessment by VAS score and blood samples and CEA dosage. The use of concomitant treatment or drugs for pain control will be recorded. Adverse events/complication will be noted.After clinical evaluation, the patients will be submitted to rectoscopy, colonoscopy, MRI, pain assessment, blood samples and CEA dosage as above. The use of concomitant treatment or drugs for pain control will be recorded. Adverse events/complication will be noted.After clinical evaluation, the patients will be submitted to rectoscopy, colonoscopy, CT, MRI, clinical evaluation, pain assessment, blood samples and CEA dosage as above. The use of concomitant treatment or drugs for pain control will be recorded. Adverse events/complication will be noted.The response of treatment in both the control arm and the treatment arm will be assessed with the histopathological TRG on tissue specimens after local excision.The total number of cases with TRG 1 and 2  will be If the histological examination documents the presence of carcinoma with at least one of the following characteristics: 1. ypT ≥ 2; 2. Positive margins; 3. TRG > 2; 4. Poorly differentiated form (G3), the local excision will be considered inadequate treatment and the patient will undergo TME.Any Adverse Event (AE) will be recorded on the case report form as well as the intercurrent events. Subjects will be examined at each visit or if the subject reports any unusual symptoms. The numbers and severity of such incidents will be included in the statistical analysis. All Adverse events (AEs and SAEs) will be recorded on the AE form. Serious adverse events (SAEs) will be reported to the IRB. Toxicity will be assessed using the National Cancer Institute’s Common Toxicity Criteria for Adverse Events, version 5.0 (CTCAEv5) .A collection of adverse events will be conducted at Visit 2 to individuate the AE rate due to neoadjuvant treatment. Neoadjuvant therapy is usually well tolerated, although some patients can require hospital admission for the management of diarrhea or significant late small bowel toxicity, attributable to radiotherapy ,35,36,37A following collection of adverse events will be conducted at Visit 3, before LE, to individuate the AE rate due to added treatment (ECT).The sample size was determined by the Group-Sequential Superiority approach with a Margin Tests for the Difference of Two Proportions  using a The proportion in the treatment group is assumed to be 0.25 under the null hypothesis and 0.50 under the alternative hypothesis. The proportion in the control group is assumed to be 0.25. The significance level of the one-sided test was targeted at 0.10 and the power at 80%.An interim analysis will be conducted when 28 patients are enrolled (the percent of the sample size accumulated up to the corresponding look will be 40.0%). If a complete response rate to the TRG after local excision is noted in less than 25% of patients in the treatment group and/or feasibility/safety was not demonstrated, the study will be discontinued, otherwise a further 42 patients will be enrolled in the study.The treatment feasibility/safety of surgery following electrochemotherapy will be assessed considering the number of participants with compromised surgery following electrochemotherapy assessed by the R1 resection rate, circumferential resection margin (CRM) involvement, non mesorectal resection plane, and post-operative complications according to the Clavien-Dindo Classification. A surgical complications rate of 30–40% was reported in the literature for conventional neoadjuvant treatment followed by TME . The evap < 0.10. A Chi square test will be performed to assess statistically significant differences among the groups for the categorical variable.Complete response rate (TRG 1 and 2), disease recurrence rates and percentages will be compared among the two groups. A one-sided Fisher exact test will be performed to test the differences between the groups. Differences will be considered significant at The time interval between the recruitment date and local disease progression will be represented using the time of treatment failure and median and minimum–maximum values will be considered.The scores obtained by the QOL, pain and satisfaction questionnaires will be evaluated. Any serious adverse events and their intensity, according to the NCI-CTCAE classification (version 5.0), will be registered in the first two days post-procedure.Mean ± standard deviation will be considered for normally distributed continuous variables while the median and interquartile range (IQR) will be presented for non-normally distributed continuous variables.For continuous variables, the difference between follow-up values and baseline values will be tested by a 2-sided Student t-test or by a Mann–Whitney test while, for the categorical variables, the McNemar test and the marginal homogeneity test will be use ,50.Time to event outcomes will be analyzed using the Kaplan–Meier method and survival curves will be compared using the log-rank test.Mixed effect regression models will be used to analyze longitudinal data by adjusting for proper covariates.Interim analyses will be fixed after the first step of the study. A conclusive analysis will be performed at the end of the treatment of patients enrolled in the second stage. If a complete response rate to the TRG after local excision is noted in less than 25% of patients in the treatment group, and/or feasibility/safety was not demonstrated, the study will be discontinued. A difference of 20% for both AE rate and surgical complications rate between the two arms will be considered significant enough to discontinue the trial.The efficiency and safety of ECT have been demonstrated in preclinical and clinical Phase I and I/II studies in patients with melanoma, head and neck squamous cell carcinoma, Merkel cell carcinomas, basal cell carcinoma and adenocarcinoma nodules. The efficacy of ECT has already been proven in skin tumors, and in mucosal and submucosal tissues with high tumor control. Recent studies have demonstrated the feasibility and safety of ECT in the treatment of deep tumors  ,24,25,26https://clinicaltrials.gov/ct2/show/NCT03040180, accessed on 10 April 2021) [Moreover, colorectal cancer as well as gastric and esophageal tumors have been treated with endoluminal electrodes without adverse events or complications demonstrating a tumor regression (il 2021) .Currently, 45–55% of rectal cancer patients receive preoperative chemo-radiotherapy for LARC. TME is the gold standard in rectal cancer surgery and is comprised of resection of the rectum together with the fatty tissue surrounding the rectum ,60,61,62The high morbidity and functional complications associated with TME motivate the search for conservative treatments with sphincter preservation for patients with early rectal cancer at diagnosis and patients with significant tumor regression after pCRT, with the advantage of reducing morbidity and providing a “true” organ-sparing approach . It has Complete pathological response after neoadjuvant therapy in rectal cancer has been shown to be an independent predictor of overall survival and a decrease in local recurrences; up to 30% of patients will achieve a complete or near complete response ,47,48.The idea is to use electrochemotherapy in patients with LARC and major clinical response after neoadjuvant therapy, before surgery  and assuming an increase in the local control of residual disease and of complete response rate, confirmed by histopathological examination using Tumor Regression Grade. The TRG is a powerful indicator of response to a neoadjuvant treatment in rectal cancer and can measure the effectiveness of neoadjuvant therapy.ECT has been shown to have limited side effects, a high degree of patient acceptability and acceptable costs. Treatment is performed under local or general anesthesia and does not always require hospitalization. The outcome is to verify the feasibility/safety of electrochemotherapy in this indication and the increase of complete response rate in the treatment group with respect to control group.This study is a phase IIb controlled, randomized, clinical trial conducted in order to evaluate the increase in the complete response rate after neoadjuvant treatment followed  or not (control group) by ECT in LARC and to spare organ function due to total mesorectal excision (TME).


Household air pollution (HAP) is associated with adverse human health impacts. During COVID-19 Lockdown Levels 5 and 4 (the most stringent levels), South Africans remained at home, potentially increasing their exposure to HAP.To investigate changes in fuel use behaviours/patterns of use affecting HAP exposure and associated HAP-related respiratory health outcomes during COVID-19 Lockdown Levels 5 and 4.This was a cross-sectional online and telephonic survey of participants from an existing database. Logistic regression and McNemar’s test were used to analyse household-level data.Among 2 505 participants, while electricity was the main energy source for cooking and heating the month before and during Lockdown Levels 5 and 4, some households used less electricity during Lockdown Levels 5 and 4 or switched to “dirty fuels.” One third of participants reported presence of environmental tobacco smoke in the home, a source of HAP associated with respiratory illnesses. Prevalence of HAP-related respiratory health outcomes were <10% (except dry cough). Majority of households reported cooking more, cleaning more and spending more time indoors during Lockdown Levels 5 and 4 – potentially exposed to HAP.Should South Africa return to Lockdown Levels 5 or 4, awareness raising about the risks associated with HAP as well as messaging information for prevention of exposure to HAP, including environmental tobacco smoke, and associated adverse health impacts will be necessary. On March 11, 2020, the World Health Organization (WHO) declared the outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel coronavirus disease (COVID-19), a pandemic 14. In SouOn March 26, 2020, South Africa went into National Lockdown (Alert level 5) indicating “high COVID-19 spread with low health system readiness.” [In South Africa, about 14.5 million people rely on solid or “dirty” fuels  as their main source of energy for cooking . The typFew studies have considered household air pollution (HAP) risks in lockdown periods during the COVID-19 pandemic. A Spanish study found that mean daily HAP levels increased due to the lack of suitable ventilation and more intensive cleaning using harsh cleaning products and disinfectants . Given tThe study was designed as a retrospective study based on an online/telephonic survey questionnaire. The survey was conducted in four South African provinces, namely Gauteng, Western Cape, KwaZulu-Natal, and Eastern Cape. These provinces were selected for having the highest COVID-19 cases at the time of study preparation . The stuStudy participants were drawn from a market research company’s panel. Participants were passed through a double opt-in process that confers to the European Society for Opinion and Market Research (ESOMAR) guidelines . This waA short introduction (information sheet) was given at the start of the survey. After written or verbal consent was given for online survey or telephone interview, respectively, the survey/interview was initiated.The questionnaire was designed on the lines of the questionnaire developed in the UK for national longitudinal cohort studies , bearingTable 1) namely: Part A: general participant information; Part B: socio-economic-demographic information; Part C: HAP indoors: the month before Lockdown Levels 5 and 4 , and during Lockdown Levels 5 and 4 ; Part D: respiratory symptoms/illnesses during Lockdown Levels 5 and 4; Part E: any presence of COVID-19 test/case in the household during Lockdown levels 5 and 4; and Part F: agency/choice and health promotion for informed decision-making and lifestyle changes. Finally, participants were thanked for completing the questionnaire and informed that there are health risks linked to being exposed to HAP, especially when cooking with coal, wood, or paraffin indoors. Moreover, if they had any concerns, or wanted more information, they were advised to contact their healthcare providers.The questionnaire was divided into six parts (A Living Standards Measure (LSM) which is a means of segmenting South African people according to their living standards, where 1–4 equate to the lowest, 5–7 intermediate and 8–10 the highest LSM status was known from data in the market research company’s panel and also included in the dataset.To account for the different fuel use patterns across the four provinces (heating with solid fuels versus electricity) , it was Data were converted from a .txt file to Stata version 16.1 . DemograTo meet objective 1, changes in fuel use patterns the month before and during Lockdown Levels 5 and 4 were examined using the generalized McNemar’s test, to account for both discordant as well concordant pairs i.e., participants who experienced changes in their fuel use as well as participants who have not changed their fuel use patterns. P-values < 0.05 were considered statistically significant.To meet objective 2, respiratory symptoms were compared for the month before and during Lockdown Levels 5 and 4 using logistic regression.Objective 3  was discussed in light of the findings from objectives 1 and 2 in the discussion. No analyses were done stratified by province since this was an overall analysis.Table 2 provides the sample descriptive statistics by questionnaire variable. Majority of participants were Black African and female. About 40% of participants were from Gauteng likely since this is the most populous province in South Africa. There is also free Wi-Fi in certain public spaces of major cities, e.g., Tshwane Free Wi-Fi in Pretoria, possibly making it easier to complete the survey, compared for example to the Eastern Cape province where Internet access may have been a challenge. In this instance, CATI was applied to try and include more participants from areas without internet access.The survey was conducted between October 29, 2020 and December 2, 2020. Table 3). Patterns of HAP exposure factors, excluding fuel use types for cooking and heating, were similar based on self-reported responses for questions pertaining to the month before and during Lockdown Levels 5 and 4. Most participants (about two-thirds) reported naturally ventilating their homes and did not have presence of mould, animals, incense burning or environmental tobacco smoke in their dwellings both before and during Lockdown Levels 5 and 4. However, about one in three participants did report presence of one or more of these HAP exposure factors. This being a particular concern for environmental tobacco smoke which is known to contribute to HAP and adverse respiratory health outcomes.Overall, electricity was the most commonly used energy source for cooking and heating followed by gas (Table 4). The main contributions were due to changes from electricity to gas  and gas to electricity .Changes in fuel use patterns for cooking for the month before versus during Lockdown Levels 5 and 4 were not statistically significantly different (Table 5) though the actual numbers are small. The contributions were due to changes from coal to electricity , electricity to coal , electricity to gas , gas to electricity , electricity to paraffin  and paraffin to electricity .Changes in fuel use patterns for heating was significant (Table 6) when less of the health outcomes were experienced during the latter.Self-reported prevalence of doctor or nurse-diagnosed health outcomes was generally low at less than 10%, except for dry cough. There were statistically significant differences in the responses for dry cough, wet cough and hay fever for the month before versus during Lockdown Levels 5 and 4 (One in three participants reported someone in the household having had a COVID-19 test during Lockdown levels 5 and 4. One in five respondents reported someone who had a positive COVID-19 test in the household during Lockdown levels 5 and 4.Table 7). More than 80% of participants reported finding it difficult to make financial ends meet.Majority of households reported cooking more, cleaning more and spending more time indoors during Lockdown Levels 5 and 4 compared to the month before Lockdown Levels 5 and 4  during Lockdown Levels 5 and 4.From our study findings, electricity was the most commonly used energy source for household cooking and heating the month before and during Lockdown Levels 5 and 4. However, there was a slight (non-significant) change in electricity use for cooking with some (relatively few) households using electricity less during Lockdown Levels 5 and 4 compared to before Lockdown Level 5 . This could be due to 83% of respondents reporting to struggle to make financial ends meet during Lockdown Levels 5 and 4, and possibly switching to alternate energy sources , as wellWhile most participants (about two in three) reported naturally ventilating their homes and did not have presence of mould, animals, or incense burning  in their dwellings both before and during Lockdown Levels 5 and 4, of particular concern was environmental tobacco smoke being present in one in three households both before and during Lockdown Levels 5 and 4. Environmental tobacco smoke is a contributor to HAP and is known to impact respiratory health especially among children, causing asthma, infection and wheeze 19.In terms of objective 2, we found that self-reported prevalence of doctor or nurse-diagnosed respiratory health outcomes were less than 10% for both the month before and during Lockdown Levels 5 and 4. This could be as a result of recall bias, or be indicative of the Lockdown and health-seeking behaviours. A study in KwaZulu-Natal province showed no difference in daily clinic visitation at ambulatory clinics, suggesting that citizens continued to seek medical care during lockdown as done before Lockdown Level 5 . HoweverIn terms of household behaviours, we saw majority of households reported cooking more, cleaning more and spending more time indoors during Lockdown Levels 5 and 4 compared to the month before. A study in rural Odisha, India, reported 41% of respondents having changed their household cleaning practices as a result of the COVID-19 outbreak or Lockdown . Of thesWith respect to objective 3, if we consider whether fuel use patterns for heating and cooking, and other behaviours related to HAP exposure changed during Lockdown Levels 5 and 4, we have insufficient evidence to confirm any significant changes, besides small changes in fuel type used for heating  and more cooking and cleaning.We acknowledge that this was a survey-based questionnaire that relied on self-reporting hence there was a risk of recall (or response) bias. We tried to address this bias by requesting that participants report on doctor or nurse-related health symptoms and diseases and not self-reported symptoms/illnesses that may be more easily forgotten. We also tightened the time frame for occurrence of health symptoms and diseases to the month before and during Lockdown Levels 5 and 4 which were difficult periods in everyone’s lives and more likely recalled.This study was conducted using non-probability quota sampling which does entail sampling bias. We set quotas for different types of respondents, mainly dependent on their home province, with more people surveyed from provinces affected by COVID-19 (at the time of project set-up) being surveyed. The aim of quota sampling was to control the composition of the final achieved sample at the design stage already. We did conduct the survey using probability sampling using a sample frame, and therefore there were no “non-responses” for which to account. A different sampling approach may be useful to apply in the future with a focus on ensuring good coverage of all LSM groups since we had few participants from the low LSM groups.Self-reporting happened through the telephonic interviews as well as the online self-complete interviews. This is a limitation because of the current pandemic that requires contactless interviewing methods rather than face-to-face. The number of questions that one can have in an online or CATI survey is also restricted based on previous experience from the company who assisted with the data collection. This limited the questions we were able to ask and therefore prohibited some analyses, for example, comparing an individual who said they had COVID symptoms and whether or not they had a COVID test, followed by whether it was positive or negative. Future studies, preferably face-to-face, would permit for longer surveys where more detailed information would be gathered about all individuals separately living in the household.Due to budget constraints and the cost of CATIs, the sample was skewed to the online survey which included a higher proportion of people from middle-to-high income groups. This affected the proportion of households using fuels associated with HAP, i.e., not electricity. Future studies should consider spending more funds on CATIs to gather data from lower-income households more likely to be exposed to high levels of HAP from the burning of solid or “dirty” fuels.Our survey questionnaire did not ask about the urbanicity of the participant’s dwelling, i.e., urban, rural, peri-urban, peri-rural etc. and this should be included in future surveys. We also did not ask about specific type of geographical zone, such as township, suburb, inner city etc. and this information would be useful in future studies.While several of the HAP respiratory health outcomes are similar to those for COVID-19, the questions about whether anyone in the household had had a COVID-19 test and any positive cases of COVID-19 in the household were included to consider presence or absence of COVID-19 in households. Future studies, with questions about specific HAP-related respiratory outcomes and specific COVID-19 health outcomes, as well as associated risk factors, might be useful if one wanted to assess the impact of HAP exposure on HAP-related respiratory outcomes and also to assess the impact of HAP exposure on COVID-19 specific symptoms/ill health effects.Should South Africa return to Lockdown Levels 5 and 4 (and perhaps even less stringent levels given the economic climate of the country) efforts will need to be made to raise awareness about the risks associated with HAP exposure, especially from the burning of “dirty” fuels for heating. Moreover, raising public awareness of the risks associated with environmental tobacco smoke is required. Future research should focus on groups most vulnerable to HAP exposure including people living in poverty, people living in townships, children, the elderly and people with pre-existing diseases.


Functional coatings based on the assembly of submicrometric or nanoparticles are found in many applications in the biomedical field. However, these nanoparticle-based coatings are particularly fragile since they could be exposed to cells that are able to internalize nanoparticles. Here, we studied the efficiency of RAW 264.7 murine macrophages to internalize physisorbed silica nanoparticles as a function of time and particle size. This cell internalization efficiency was evaluated from the damages induced by the cells in the nanoparticle-based monolayer on the basis of scanning electron microscopy and confocal laser scanning microscopy observations. The internalization efficiency in terms of the percentage of nanoparticles cleared from the substrate is characterized by two size-dependent regimes. Additionally, we highlighted that a delay before internalization occurs, which increases with decreasing adsorbed nanoparticle size. This internalization is characterized by a minimal threshold that corresponds to 35 nm nanoparticles that are not internalized during the 12-h incubation considered in this work. Functional coatings based on the assembly of submicrometric or nanometric particles (NPs) are found in many applications in biomedical field. For example, it was recently proposed that NPs-based monolayers or multilayers can serve as drug delivery platforms in which physisorbed NPs are loaded with drugs for precise drug delivery and prolonged drug release ,3,4,5,6.NPs-based coatings found on biomaterials such as catheters, prostheses or nanoparticle-based biosensors can be exposed to cells when implemented. As central actors of the early immune response, macrophages are likely to be recruited on site soon after implementation. During their migration, these cells might develop interactions with the biomaterial surface and try to internalize the NPs. These interaction forces, which can be as high as a few micronewtons ,12,13, cThis information about the stability of NPs-based coatings exposed to cells raises the fundamental question concerning the cell uptake efficiency of adhering NPs or bacteria, which was only recently addressed in the literature ,19,20,21In this work we have studied efficiency of RAW 264.7 murine macrophages to internalize silica particles physisorbed in the form of a monolayer through weakly attractive van der Waals interactions and electrical double-layer interactions. The novelty of our work lies in the study of a wide particle size range, from 35 nm to 450 nm, and short successive incubation times: 1 h, 3 h, 6 h, 9 h and 12 h, in order to acquire insights in the particle size dependence and time dependence of the internalization process. The cell internalization efficiency was evaluated from the damages induced by the cells in the nanoparticle-based monolayer on the basis of scanning electron microscopy and confocal laser scanning microscopy observations.Nanoparticles monolayer fabrication. Nanoparticles (NPs) monolayers consisted in fluorescent silica NPs functionalized with carboxyl (−COOH) head groups, that were physisorbed on amine (−NH2) functionalized silicon wafers. For this, single side polished Si wafers  were divided into 1 cm × 1 cm samples. They were first cleaned in a piranha solution and functionalized with aminopropyltrimethoxysilane  by immersion for 12 h in a 2 mM ethanol solution. After rinsing with ethanol, the samples were dipped in suspensions of fluorescent silica NPs functionalized with carboxyl (−COOH) head groups  at a concentration of 25 mg/mL for 2 h. Samples were then removed from the NPs suspension and rinsed with deionized water three times. It is important at this step to avoid drying of the surface. Samples were then stored in 70% ethanol in water solution for further use.Cell culture. Murine macrophage-like RAW 264.7 cells  were cultivated in DMEM (Sigma-Aldrich) supplemented with 10% inactivated fetal bovine serum , 20 mM HEPES (Sigma-Aldrich) and 2 mM L-glutamine (Sigma-Aldrich) in a humidified incubator at 37 °C and 5% CO2. Cells were routinely passaged maximum up to 20 times. Cells were detached once they reached 80–90% confluence using Accutase® , following the procedure provided by the supplier. The cell concentration was then adjusted for further use.Exposure of the NPs-based monolayers to the cells. Ethanol was removed from the silica wafers supporting the NPs monolayer by three successive washes in the cell culture medium. The samples were then placed in a 24-well culture plate and immediately covered with 1000 µL of a cell suspension at a density of 2 × 104 cells/mL. It is important at this step to avoid drying of the particle monolayers. The cells were then fixed (as described below) after different incubation times for scanning electron microscopy and confocal fluorescent microscopy observations.Scanning electron microscopy (SEM). Before SEM observation, the cells were fixed following a standard procedure. All drying steps were performed in a 24-well culture plate containing the samples. The culture medium was first replaced with PBS to rinse the sample. PBS was then replaced with a glutaraldehyde (4%)/paraformaldehyde (2%) solution in PBS. Cells were then dehydrated by replacing the glutaraldehyde/paraformaldehyde solution successively with an alcohol gradient of 30%, 50%, 70%, 80%, 90% and 100% (twice), each time for 10 min. All the previous steps were performed while avoiding drying of the samples. Finally, the 100% ethanol solution was replaced by hexamethyldisilazane  for 10 min. After drying under air flow in a hood, the samples were sputter coated with gold and observed using a Philips XL-30 FEG scanning electron microscope at 5–15 kV in SE mode.Confocal laser scanning microscopy (CLSM). Cells, either live or fixed, were observed using an upright confocal laser scanning microscope (CLSM) system LSM 700 . For live cell observation, cells were detached following the procedure described earlier and stained with CellTracker Red CMTPX  following the protocol provided by the manufacturer. Observation was performed using an incubator chamber mounted on the microscope stage and a 20×/1.0 plan apochromat water immersible objective lens . In other experiments, cells were fixed in 4% paraformaldehyde/PBS  for 15 min, permeabilized with 0.1% Triton X100  for 15 min and finally stained with Hoechst 33258  for observation of the nucleus and with Texas Red phalloidin  to visualize the cell body. Fixed cells were observed using a 20×/1.0 plan apochromat water immersible objective lens .Evaluation of the internalization efficiency. The internalization efficiency was evaluated on the basis of the damaged surface area, the percentage of particles removed from the damaged area and the number of particles internalized. The damaged surface area was considered around an isolated cell, as the area showing missing particles compared to the virgin particle monolayer. Whereas the borders of the damaged areas area are obvious in the case of the largest particles, these borders were more roughly evaluated by eyes in the case of the 100 nm and 50 nm particles. The percentage of the particles removed from the damaged area was evaluated by counting the particles left in the damaged area (either using ImageJ or by hand) and normalizing by surface density of the particles in the virgin monolayer. This thus simply led to the number of particles internalized by one cell.Statistics and reproducibility. We have considered 8 to 10 cells for a given NPs size and incubation time. The whisker lengths of the box charts are 1.5 time the inter-quartile range before corrections , and only the outliers are represented. Since the data is not normally distributed, we used the Mann-Whitney test to calculate p values. The trends discussed in this article were reproduced in an independent experiment  [We prepared model surfaces consisting of monolayers of fluorescent silica nanoparticles (NPs) adsorbed on silicon wafers. The amorphous silica NPs were functionalized with carboxyl groups (see ctively) . The NPsThe damages induced by the RAW 264.7 macrophage in the nanoparticle-based monolayers were evaluated on the basis of scanning electron microscopy observations (SEM) after fixing the cells. Such damages were already observed after 24 h incubation by Böcking et al. in 200 nm large silica particles multilayers exposed to C2C12 cells , and by In a first approach, the damages in the monolayers can be quantified through the evolution with time of the damaged surface area around the cells. The graph in p < 0.05 between each pairs of data). This result is characterized by an extremely high percentage for larger NPs (450 nm and 300 nm), equal to and near 100%, respectively, as shown in The damages in the nanoparticle-based monolayers can also be evaluated by the percentage of NPs removed from damaged area. Note that in the SEM images  the damaThe damage to the nanoparticle-based monolayer can be directly related to the cellular internalization process. Indeed, each surface presenting a lack of NPs is associated to one cell, as we can observe on Therefore, the cell internalization efficiency can be evaluated in a first step by converting the percentage of the NPs removed from the damaged area b into a These two distinct size-dependent behaviors fit those usually associated with cell internalization processes. Indeed, the fact that 450 nm and 300 nm NPs lead to the most efficient internalization is in agreement with the characteristic sizes for which the active phagocytosis mechanism is observed. These large NPs are expected to be internalized by macrophages following an active phagocytosis process based on the activation of the actin-based machinery ,27,28,29This result is also in agreement with the work of Septiadi et al. in which physisorbed silica particles (480 nm large) are detached and internalized by primary human monocyte-derived macrophages following a phagocytic process . They rep-values indicated in The cell internalization efficiency can also be evaluated considering the number of NPs internalized by one cell as a function of time a. As expThe number of NPs internalized by one cell as a function of time a does noIn the case of small particles (from 200 nm to 35 nm), SEM observations have shown complete nanoparticle-based monolayers, without any damages, even after many hours of incubation. This delay before the clearing occurs was shown to increase with decreasing the size of the particles. Silica NPs in the monolayer adhere indeed to the substrate through both weakly attractive van der Waals interactions and electrical double-layer interactions . Simple Even though mostly described in confined environments, this result on smaller NPs might be explained by an amoebial migration mode, in which cell migration occurs without pulling the substrate, but following an amoeba-like behavior, with expansion and contraction of the actin cortex ,41,42,43In the hypothesis of an endocytic uptake suggested earlier, it was shown that engulfment forces can reach from tenths to hundreds of pN ,45. In tTo summarize, physisorbed silica nanoparticle-based monolayers were exposed to RAW 264.7 macrophages. Based on SEM observations, particle internalization by cells from the monolayers was analyzed at short incubation times  and for six different particle sizes . This new approach based on short incubation times and a wide range of particle sizes gave insights in the internalization kinetics of physisorbed particles.We thus clearly observed two distinct internalization regimes. One regime observed for large particles (450 nm and 300 nm) in which almost all the nanoparticles are removed and internalized by the cells. A second regime observed for smaller nanoparticles , in which only a part of the particles is removed by the cell, with a percentage of particles removed that decreases with decreasing the size of the particles. These two regimes were associated to phagocytosis and endocytosis, respectively, as suggested recently by previous studies. This approach also revealed a delay before internalization, that increases with decreasing the size of the NPs. Otherwise stated, this delay corresponds to a time windows for which the nanoparticle-based monolayers are stable under adhering and crawling macrophages. Finally, we observed that 35 nm large nanoparticles were not internalized by the macrophages even after 12 h of incubation.Considering adsorbed particles exposed to cells reveals cellular internalization behaviors that are not observable in studies where cells interact with NPs in solution. The influence on the internalization process and efficiency of parameters such as geometrical constraints or NPs adhesive force can actually be explored in this way. Such experimental approach shed light on both fundamental cell behaviors and interactions between cells and materials textured with NPs, opening new research perspectives relevant to biomaterials, biosensors and drug delivery strategies.


The emergency department (ED) setting might be a suitable environment for conducting interventions for smoking cessation. The present study aims to determine the feasibility and effectiveness on smoking cessation of 5A’s counselling administered to ED patients by nurses.Parallel group randomized trial assessing 5A’s counselling for smoking cessation vs. usual care at a University Hospital in the North of Italy. The primary end-point was prevalence of tobacco-free patients. The secondary outcomes at 6- and 12-month follow-up were (i) consecutive past 30-day smoking abstinence; (ii) past 7-day 50%, or more, decrease in daily tobacco consumption over baseline; and (iii) number of attempts to quit smoking.n = 262) or usual care (n = 218). Intention to treat analysis displayed no differences in primary and secondary outcomes between groups. A slight but not statistically significant enhancement in cessation was recorded in the intervention group  at 6 months, whereas a reversed observation at 12 months . Similar results were obtained for the secondary outcomes. Per protocol analysis increased the size of the results. Of the 126 smokers receiving counselling, 18 were visited and treated at the local smoking cessation centre, with 12 of them successfully completing the treatment.A total of 480 patients were randomized to intervention (The results of this study indicate that the ED is not a suited environment for 5A’s counselling.  Smoking is a major public health issue worldwide. This is particularly worrisome in Italy, where 25.7% of adults (18–69 years old) currently smoke tobacco.Moderate-quality evidence suggests that brief intervention performed by nurses to promote and support smoking cessation can increase the number of people achieving prolonged abstinence.The five major components of 5A’s smoking cessation intervention in the healthcare setting are the following: (i) ask every patient about tobacco use; (ii) advice current smokers to quit; (iii) assess the patient’s motivation to quit; (iv) assist both patients who want to quit and patients who are not interested in quitting using motivational interventions; and (v) arrange the follow-up schedule.Among emergency department (ED) patients, there is a higher prevalence of smokers compared to the general population (21–48%).In the last two decades, several large-N studies investigating the feasibility and efficacy of counselling for smoking cessation during ED stay have failed to demonstrate a statistically significant increase in smoking quit rates in patients receiving counselling at 3-,Although a systematic review has recently shown a stronger impact of smoking cessation interventions conducted in EDs compared to other healthcare settings, it did not discriminate the role of specific counselling from that of the health visit itself.ad hoc self-administered questionnaire at baseline. The protocol registration number is: NCT04107753.The study was a parallel group RCT with an allocation ratio of 1:1. The study was entirely conducted at the ED at ‘Maggiore della Carità’ University Hospital, Novara, Italy. Eligible patients were randomly assigned to intervention (i.e. 5A’s counselling) or control arm . The patients’ smoking habits were assessed by means of an The study was approved by the Ethical Committee of the University Hospital of Novara on 7 January 2016 (n. 602/CE).The study population included individuals aged 18 years old or older admitted to the ED from April 2017 to May 2018, self-declared as ‘current smokers’, who gave their written informed consent. Exclusion criteria were (i) an emergency condition labelled with a red priority code according to the Italian triage protocol; (ii) assignment of the patient to a nurse not previously trained in 5A’s counselling; (iii) a mental illness or a history of psychiatric illness; (iv) inability to understand either written or oral Italian; and (v) ongoing treatment for tobacco addiction.,For sample size calculation, we assumed a cessation rate in the general population of 8.4%,From April 2017 to May 2018, as part of the triage evaluation, all patients accessing the ED were asked about their smoking habits. Smokers were then proposed to participate in the study. Once the input of the clinical data and the oral consent were obtained from the patients, these latter were randomly allocated to the control or intervention arm by the hospital management software and were given the clinical documents needed for the visit together with the study informed consent sheet. A printed univocal mark at the bottom of this informed consent allowed the healthcare providers to identify those patients enrolled in the study according to their allocation. Enrolled subjects were blinded to the assigned arm. Patients who gave written information consent were also asked to fill out a questionnaire aimed to investigate the characteristics of their smoking habits.A dedicated operator, not part of the ED staff, took care of all aspects pertaining to the collection of the informed consents and questionnaires and acted as liaison between the nurses and the patients in the intervention group.,Centro per il Trattamento del Tabagismo (CTT) , inside the Department of Addiction of the Novara’s Local Health Unity in order to plan intensive counselling. Instead, patients not motivated to quit were only administered motivational counselling.The intervention group received a brief counselling based on a 5A’s model  partly aAll patients of the intervention group received a card containing information on the CTT, a few dedicated website links and the telephone number and location of the CTT. Motivated patients were referred to the CTT, and, within a few days from the ED visit, these patients were contacted by a CTT operator in order to schedule their first meeting.The brief intervention was carried out by the nurse who took clinical care of the patient. All the nurses acting as ‘intervention providers’ received specific training from the CTT staff. This training was specifically focused on the description and application of the 5A’s intervention, the importance of the relationship with patients and the acquisition of specific skills through role play techniques. The training session was conducted a few months before the beginning of the enrolment and consisted of 2 h of frontal lesson and 2 h of role-playing exercises. No re-training courses were scheduled.Control patients received the ‘usual care’, which consisted in advising them to quit without conducting structured counselling.The actual implementation of every step of the ‘intervention as expected’ was recorded in a process checklist. It included information on the implementation of tasks and the reasons for lack of implementation. Finally, at the end of the recruitment phase, an anonymous survey was administered to the nurses involved in the study to address barriers to implementing 5A’s counselling.The primary outcome was the point prevalence of tobacco-free patients  both at 6 and 12 months from the intervention.The secondary outcomes were (i) consecutive past 30-day smoking abstinence at 6- and 12-month follow-up; (ii) past 7-day 50%, or more, decrease in daily tobacco consumption over baseline at 6- and 12-month follow-up; and (iii) the number of attempts to quit smoking at 6- and 12-month follow-up. All the information was self-reported.The follow-up was performed at 6 and 12 months through computer-assisted telephone interview. In order to minimize dropouts, three call attempts were made before considering the patient as lost to follow-up.t-tests for continuous variables. Absolute (N) and percentage (%) frequencies were reported for the categorical variables, while mean and standard deviation were reported for the numeric ones.Demographic and clinical characteristics of treatment and control groups were compared to assess the balance across the experimental arms at baseline using chi-square for categorical variables or The outcomes were dichotomized, and RRs for the expected outcomes, with their 95% confidence intervals (CIs), were obtained by comparing the experimental with the control group. The effects of the treatments were evaluated using intention-to-treat (ITT) analyses. A sensitivity analysis using the per protocol (PP) approach was performed to evaluate the possible role of the intervention among adherent patients.® software, thereby ensuring confidentiality and high privacy level according to the Italian law. All statistical analyses were performed using SAS software .All data were recorded through RedCapGiven the low level of adherence to the study procedures, a questionnaire addressing barriers to the implementation of the intervention was prepared and administered to all nurses involved in the study. This was later analysed in a descriptive way. For numeric variables, median and interquartile ranges (IQR) were used.From April 2017 to May 2018, 890 smokers admitted to the ED of the Novara University Hospital were asked to participate in the study. Among these patients, 293 (32.9%) refused to participate, while 597 (67.1%) gave their verbal informed consent and were thus randomized. However, of these latter, only 480 subjects (80.4%) filled out the baseline questionnaire and were thus included in the study, which comprised 262 subjects randomized to the intervention group and 218 subjects allocated to the control group.In the intervention group, 126 out of 262 subjects (48.1%) received smoking cessation counselling, with 91 of them (72.2%) accepting to be contacted by the CTT and 18 starting a treatment at the CTT .N = 295, 61.5%), with a mean age of 43.7 ± 14.8 years. The most frequently assigned priority code at triage was green . Furthermore, only a limited number of patients were admitted to the ED for a potential smoking-related condition: 16.8% (n = 44) in the intervention group and 17.4% (n = 38) in the control group. The mean daily number of cigarettes smoked did not vary between the two study groups, with most of the patients showing a low-to-moderate dependence according to the Fagerström test score.The patients enrolled in the study were predominantly males  of them were actually contacted and 37 accepted to present themselves at the CTT to start a treatment. The other 24 declared to be not sufficiently motivated or not interested, or to have already quit. Among those having accepted, however, only 18 subjects actually attended the first visit and started the treatment program. Notably, 6 of these subjects were loss to follow-up, while the remaining 12 (66.7%) completed the treatment and were smoke-free—according to hair analysis and expiratory Carbon moxodine tests—at subsequent follow-ups. The data relative to the intervention process, available for 101 out of 136 patients randomized to the intervention group who not received the smoking cessation counselling, provided an explanation for the non-adherence. While in 41.9% of cases counselling had not been administered due to organizational issues, such as time constraint, 34.6% of patients had refused the treatment. In the remainder of cases the survey had not been filled out.All 37 nurses originally trained for the study were invited to participate in a post-study survey, but only 19 (51.4%) of them completed it. The responders estimated to have screened a median of 50 subjects (IQR 18–130), to have enrolled a median of 6 patients (IQR 5–14) and to have performed a median of 5 interventions (IQR 5–20) during the whole study period.The median self-reported feasibility level of the complete study procedure—from enrolment to treatment—was 38 (IQR 18–59). The main issues reported were time restrictions (73.7%), high refusal rate after screening (68.4%) and high number of patients withdrawing their consent (11%).Overall, even though nurses regarded 5A’s counselling as being important (median 50 with IQR 30–74), they considered such intervention as not feasible in the ED setting . Among those answering the questionnaire, five nurses (26.3%) were current smokers, whereas the majority of them stated that individual smoking habits did not affect the way they counselled patients.This RCT investigated the feasibility and effectiveness of 5A’s brief and structured counselling administered by trained nurses, an intervention aimed to promote smoking cessation in Italian smokers admitted to the ED.,The ITT analysis did not detect any differences in primary and secondary outcomes between intervention and control group, both at 6- and at 12-month follow-up. Given the low rate of adherences to the protocol (<50%), a PP analysis was also carried out, revealing a slightly higher prevalence of tobacco-free patients in the intervention group at 6-month follow-up, compared to the control group, a trend that decreased at 12 months. These results are consistent with two previous studies performed in the ED setting,With regard to feasibility of 5A’s counselling, our findings suggest that such intervention should not be recommended in the ED setting for the following two main reasons:Only 126 out of 316 smokers randomized to the intervention group were actually given counselling. Furthermore, despite this very low number of candidates, the nurses were able to counsel only 40% of them. In the vast majority of cases, the nurses identified ‘lack of time’ as the main reason for not being able to administer the counselling. The unfeasibility of such intervention can therefore be ascribed to current working conditions of healthcare professionals in understaffed and overcrowded public Italian hospitals, especially EDs. Thus, in light of these findings, future efforts in promoting smoking cessation, rather than creating additional workload for ED staff, should target a wider audience by means of informative posters and brochures displayed in the ED waiting room, promoting smoking cessation and referring patients to local CTTs.The observation that 33.3% of smokers admitted to the ED refused to participate in the intervention and 11.7% did not fill out the questionnaire, together with the fact that only 12 out of 126 smokers referred to the local CTT actually completed their treatment, indicates that the ED stay is not a teachable moment for smokers, at least in this Italian setting. Indeed, it is conceivable that their high level of stress due to an overcrowded environment combined their heightened psychological distress can make ED patients less receptive to prevention messages.,,Previous experiences in different health settings showed better results than our study; e.g. the FRICC study that was conducted in dental clinics.Taken together, these observations lead us to understand that, in the future, any smoking cessation program should be developed in other clinical contexts rather than in an ED similar to our one: previous experiences showed that high accession facilities represent an optimal setting for tobacco cessation interventions but, in our opinion, our ED mismatched two major requirements: a quiet environment and enough time availability.n = 1200), only 816 subjects were screened for inclusion. In these 30 months, an estimated number of 20 000 smokers admitted to the ED of Novara could have been potentially included in the study. Thus, given the low statistical power of our study, we cannot definitively rule out that the smoking cessation counselling given to ED patients may have proven effective had we analysed a larger population.A limitation of this study is represented by the limited size of our study population. Even though the period of enrolment was extended from 5 to 14 months to reach the expected number of participants (ad hoc recruited junior researcher.Another limitation is represented by the sub-optimal screening and enrolment procedures. While these tasks should have been carried out by specifically trained ED personnel throughout the course of the study, most of them were concentrated in a few months and conducted by an In conclusion, a well-standardized intervention conducted by trained personnel in the framework of an RCT coordinated by a locally renowned university shows an extremely and disappointing low level of implementation. Moreover, the relatively low number of smokers enrolled, accompanied by a high number of non-adherents and dropouts occurring during all the phases of the study, from enrolment to adherence to the treatment and to attendance to the follow-up implies that the ED stay should not be considered as a teachable moment. Nevertheless, the high rate of smoke-free patients among those receiving the treatment in the CTT—12 out of 18 patients at the 6-month follow-up suggest that a close cooperation between CTTs and EDs may favour smoking cessation in those patients who are motivated to quit. Thus, we strongly believe that efforts should be undertaken to implement a ‘fast track’ smoking cessation plan allowing ED physicians to promptly refer ready-to-quit smoker patients to a CTT.