Overexpression of Douglas-Fir LEAFY COTYLEDON1 (PmLEC1) in Arabidopsis Induces Embryonic Programs and Embryo-like Structures in the lec1-1 Mutant but Not in Wild Type Plants.

Somatic embryogenesis (SE) is the most promising method for the quick propagation of desirable plant genotypes. However, application of SE to conifers remains challenging due to our limited knowledge about the genes involved in embryogenesis and the processes that lead to somatic embryo formation. Douglas-fir, an economically important lumber species, possesses a homolog of the angiosperm embryo-regulatory LEC1 gene. In the present study, we analyzed the potential of Douglas-fir PmLEC1 to induce embryonic programs in the vegetative cells of a heterologous host, Arabidopsis thaliana. PmLEC1 complemented the Arabidopsis lec1-1 null mutant and led to a variety of phenotypes ranging from normal morphology to developmental arrest at various stages in the T1 generation. PmLEC1 did not affect the morphology of wild type Arabidopsis T1 plants. More profound results occurred in T2 generations. PmLEC1 expression induced formation of recurrent somatic embryo-like structures in vegetative tissues of the rescued lec1-1 mutant but loss of apical dominance (bushy phenotype) in wild type plants. The activation of embryonic programs in the lec1-1PmLEC1 T2 plants was confirmed by the presence of the embryo-specific transcripts, OLEOSIN and CRUCIFERIN. In contrast, no embryo-like structures, and no OLEOSIN or CRUCIFERIN were observed in PmLEC1-expressing bushy wild type T2 plants.

Douglas-fir LEAFY COTYLEDON1 (PmLEC1) is an active transcription factor during zygotic and somatic embryogenesis.

Douglas-fir (Pseudotsuga menziesii) is one of the world's premier lumber species and somatic embryogenesis (SE) is the most promising method for rapid propagation of superior tree genotypes. The development and optimization of SE protocols in conifers is hindered by a lack of knowledge of the molecular basis of embryogenesis and limited sequence data. In Arabidopsis, the LEAFY COTYLEDON1 (AtLEC1) gene is a master regulator of embryogenesis that induces SE when expressed ectopically. We isolated the LEC1 homologue from Douglas-fir, designated as PmLEC1. PmLEC1 expression in somatic embryos and developing seeds demonstrated a unique, alternating pattern of expression with the highest levels during early stages of embryogenesis. PmLEC1 protein accumulation during seed development correlated with its transcriptional levels during early embryogenesis; however, substantial protein levels persisted until 2 weeks on germination medium. Treatment of mature, stratified seeds with 2,4-epibrassinolide, sorbitol, mannitol, or NaCl upregulated PmLEC1 expression, which may provide strategies to induce SE from mature tissues. Sequence analysis of the PmLEC1 gene revealed a 5' UTR intron containing binding sites for transcription factors (TFs), such as ABI3, LEC2, FUS3, and AGL15, which are critical regulators of embryogenesis in angiosperms. Regulatory elements for these and other seed-specific TFs and biotic and abiotic signals were identified within the PmLEC1 locus. Most importantly, functional analysis of PmLEC1 showed that it rescued the Arabidopsis lec1-1 null mutant and, in the T2 generation, led to the development of embryo-like structures, indicating a key role of PmLEC1 in the regulation of embryogenesis.

Evaluation of Adjunctive Photobiomodulation (PBMT) for COVID-19 Pneumonia via Clinical Status and Pulmonary Severity Indices in a Preliminary Trial.

PURPOSE: Evidence-based and effective treatments for COVID-19 are limited, and a new wave of infections and deaths calls for novel, easily implemented treatment strategies. Photobiomodulation therapy (PBMT) is a well-known adjunctive treatment for pain management, wound healing, lymphedema, and cellulitis. PBMT uses light to start a cascade of photochemical reactions that lead to local and systemic anti-inflammatory effects at multiple levels and that stimulate healing. Numerous empirical studies of PBMT for patients with pulmonary disease such as pneumonia, COPD and asthma suggest that PBMT is a safe and effective adjunctive treatment. Recent systematic reviews suggest that PBMT may be applied to target lung tissue in COVID-19 patients. In this preliminary study, we evaluated the effect of adjunctive PBMT on COVID-19 pneumonia and patient clinical status. PATIENTS AND METHODS: We present a small-scale clinical trial with 10 patients randomized to standard medical care or standard medical care plus adjunctive PBMT. The PBMT group received four daily sessions of near-infrared light treatment targeting the lung tissue via a Multiwave Locked System (MLS) laser. Patient outcomes were measured via blood work, chest x-rays, pulse oximetry and validated scoring tools for pneumonia. RESULTS: PBMT patients showed improvement on pulmonary indices such as SMART-COP, BCRSS, RALE, and CAP (Community-Acquired Pneumonia questionnaire). PBMT-treated patients showed rapid recovery, did not require ICU admission or mechanical ventilation, and reported no long-term sequelae at 5 months after treatment. In the control group, 60% of patients were admitted to the ICU for mechanical ventilation. The control group had an overall mortality of 40%. At a 5-month follow-up, 40% of the control group experienced long-term sequelae. CONCLUSION: PBMT is a safe and effective potential treatment for COVID-19 pneumonia and improves clinical status in COVID-19 pneumonia.

Adjunct low level laser therapy (LLLT) in a morbidly obese patient with severe COVID-19 pneumonia: A case report.

INTRODUCTION: COVID-19 poses a higher risk of complications in obese patients due to low respiratory system compliance, increased inflammatory cytokines, and an activated immune system secondary to excess adiposity. Low level laser therapy (LLLT) has significant anti-inflammatory effects and reduces inflammatory cytokines. It is noninvasive and approved for pain management and musculoskeletal injuries. Data from human and experimental animal models of respiratory tract disease suggests that LLLT reduces inflammation and promotes lung healing. CASE AND OUTCOMES: A morbidly obese 32-year-old Asian female with severe COVID-19 received four consecutive once-daily LLLT sessions via a laser scanner. Pulsed 808 nm and 905 nm laser beams were delivered over the posterior chest for 28 min. The patient was evaluated before and after LLLT by radiological assessment of lung edema (RALE) on chest X-ray, oxygen requirements and saturation, pneumonia severity indices (SMART-COP and Brescia-COVID), blood inflammatory markers (interleukin-6, ferritin, and C-Reactive protein (CRP)). Prior to treatment, oxygen saturation (SpO2) via pulse oximetry was 88%-93% on 5-6 L oxygen. Following LLLT, SpO2 increased to 97%-99% on 1-3 L oxygen. Reductions in RALE score from 8 to 3, Brescia-COVID from 4 to 0, and SMART-COP from 5 to 0 were observed. Interleukin-6 decreased from 45.89 to 11.7 pg/mL, ferritin from 359 to 175 ng/mL, and CRP from 3.04 to 1.43 mg/dL. Post-treatment, the patient noted appreciable improvement in respiratory symptoms. CONCLUSION: Following LLLT our patient showed improvement over a few days in respiratory indices, radiological findings, inflammatory markers, and patient outcomes. This report suggests that adjunct LLLT can be safely combined with conventional treatment in patients with severe COVID-19 and morbid obesity.

A 57-Year-Old African American Man with Severe COVID-19 Pneumonia Who Responded to Supportive Photobiomodulation Therapy (PBMT): First Use of PBMT in COVID-19.

BACKGROUND Coronavirus disease 2019 (COVID-19) is associated with lung inflammation and cytokine storm. Photobiomodulation therapy (PBMT) is a safe, non-invasive therapy with significant anti-inflammatory effects. Adjunct PBMT has been employed in treating patients with lung conditions. Human studies and experimental models of respiratory disease suggest PBMT reduces inflammation and promotes lung healing. This is the first time supportive PBMT was used in a severe case of COVID-19 pneumonia. CASE REPORT A 57-year-old African American man with severe COVID-19 received 4 once-daily PBMT sessions by a laser scanner with pulsed 808 nm and super-pulsed 905 nm modes for 28 min. The patient was evaluated before and after treatment via radiological assessment of lung edema (RALE) by CXR, pulmonary severity indices, blood tests, oxygen requirements, and patient questionnaires. Oxygen saturation (SpO2) increased from 93-94% to 97-100%, while the oxygen requirement decreased from 2-4 L/min to 1 L/min. The RALE score improved from 8 to 5. The Pneumonia Severity Index improved from Class V (142) to Class II (67). Additional pulmonary indices (Brescia-COVID and SMART-COP) both decreased from 4 to 0. CRP normalized from 15.1 to 1.23. The patient reported substantial improvement in the Community-Acquired Pneumonia assessment tool. CONCLUSIONS This report has presented supportive PBMT in a patient with severe COVID-19 pneumonia. Respiratory indices, radiological findings, oxygen requirements, and patient outcomes improved over several days and without need for a ventilator. Future controlled clinical trials are required to evaluate the effects of PBMT on clinical outcomes in patients with COVID-19 pneumonia.