BioOrchestra ELAVL2 / miR-485-3p target table (continued), nELAVL bullet notes, Blood section opener (WBC / PBMC / plasma table, innate vs adaptive immunity, blood sample handling)

ELAVL2 / miR-485-3p target table (continued)

	BMD-001 (ASO)	BMD-001 bind miR-485-3p	Target mi RNA of BMD-001		↑ (restore) ELAVL2 protein level	Target of ELAVL2
			↓ miR-485-3p expression	↓ the interaction between miR-485-3p and ELAVL2		SIRT1	PGC1-a	CD36	Phagocytosis	APP	Ab plaque	tau
data								- Altered CD36 - ↓ SIRT1 in AD - ↓ PGC-1alpha in AD -
IP data	안보임?				Ip p 34, 35					Ip p 34,35(APP)		p-Tau (도 10)
Non-confi			↓ (half) in 5Xfad BRAIN		(Restore) in 5Xfad BRAIN	(Restore) in 5Xfad BRAIN	(Restore) in 5Xfad BRAIN	↑ no of glial cells/plaque			↓ plaque number in DG & cortex	↓ tauC3, ↓ Tau5
Confi

• nELAVL proteins
  • are expressed exclusively in neurons in mice
  • bind numerous m RNAs
• neuronal ELAV-like proteins were shown to regulate both the alternative splicing and the abundance of these messenger RNAs.
• Y RNAs.
  • These RNA molecules do not code for proteins, and are therefore classified as non-coding RNA.
• MiRNA 란: See p7 of the patent
• ✓ PK/PD relationship in the brain for BMD-001, if they have (it seems that brain delivery is only showed by fluorescence imaging)
• ✓ Animal model? What are the endpoints?
  • Expression of miR-485-3p?
    • How much is the target microRNA increased in the brains of AD patients?
    • BMD-001 appeared to lower TauC3- and Tau5- positive tau species in primary cortical neuronal cultures. Does BMD-001 reduce brain tau load as measured by histologic staining?
    • Can the target microRNA of BMD001 be detected in biological fluids such as CSF and plasma?
      • Correlation with disease severity?
    • Moa: target mi RNA → ? → SIR1, PGC1, CD36
    • P 50. Target mi RNA → NOR, BUT what about SIR1, PGC1, CD36?
  • Target engagement
    • PET ligand?
    • miR-485-3p in CSF? Or plasma
      • Two populations of extracellular miRNAs exist in biological fluids. One can be found in vesicles such as exosomes, microvesicles, and apoptotic bodies (116, 120) while the other is associated with proteins, especially AGO2
  • Route of administration
    • Intranasal was possible?

Blood

Blood	Blood cells (45% of blood tissue)	WBC	PBMC (peripheral blood mononuclear cell) is any peripheral blood cell having a round nucleus.[1]	lymphocytes (T cells, B cells, NK cells) : Majority of PBMC	Human PBMCs Frequency (%) Monocytes 10-30% Lymphocytes 70-90% Total T cells (CD3+) 45-70% CD4+ T cells 25-60% of total CD3 CD8+ T cells 5-30% of total CD3 Total B cells 5-15% NK cells 5-10% Dendritic cells 1-2% Stem cells 0.1-0.2%
				Monocytes (monocyte 는 blood, 분화된 macrophage 는 조직내!)
				Dendritic cells
			Not pbmc	granulocytes (neutrophils, basophils, and eosinophils) have multi-lobed nuclei.
		erythrocytes		erythrocytes and platelets have no nuclei, (Not WBC, NOT PBMC )
	Platelet
Plasma (45% of blood tissue)

WBC: Neutrophils: 40% to 60%, Lymphocytes: 20% to 40%, Monocytes: 2% to 8%

	Line of Defense	Timeline	Cells	Antigen Dependency	Examples
Innate (non-specific)	First	Immediate response (0-96 hours)	Natural killer cells, macrophages, neutrophils, dendritic cells, mast cells, basophils, eosinophils	Independent	Skin, hair, cough, mucous membranes, phagocytes, granulocytes
Adaptive (specific)	Second	Long term (>96 hours)	T and B lymphocytes	Dependent	Pus, swelling, redness, pain, T and B lymphocyte response

즉, WBC 와 pbmc 는 서로 교집합을 가지되 서로 다른 category

• Wbc 의 반은 not PBMC (IE neurotrophil)!
• PBMC 는 다 WBC !

		Cell 안 보유		Cell 보유
		Serum (더 pure 하네)	plasma	PBMC	Whole blood	DNA sample	RNA sample
volume		< plasma	55% of total blood volume
composition	Same	Proteins (albumin & globulin, antibodies, hormones) O RBC X, WBC X		RBC X, WBC O	RBC O (대부분), WBC O
	difference	Fibrinogen X	Fibrinogen O, Waste removal
function		fluid	liquid
identity		what remains in the blood after it clots. a cell-free liquid that is also depleted of coagulation factors. serum is obtained after the clotting of blood, (time-consuming) Serum samples should be separated from whole blood within two hours of the blood draw	a cell-free liquid that still contains coagulation factors). plasma can be obtained before the coagulation of the blood	whole blood, diluted with PBS, is gently layered over an equal volume of Ficoll in a Falcon tube → and centrifuged for 30-40 minutes at 400-500 g without brake. → Four layers will form, each containing different cell types — the uppermost layer will contain plasma, which can be removed by pipetting Plasma / Mononuclear cells / Ficoll-Paque media / Granulocytes / Erythrocytes Density centrifugation (Ficoll-Paque) is the most typical method to isolate PBMCs. GOOD example: {Smith, 2018 #1420}	Blood begins to break down within 2 hours at room temperature, so it is important to freeze immediately or add stabilizing reagents.	QIAamp Kit from Qiagen	RNeasy
isolation, sample preparation
Used for		Blood typing, diagnostic testing	blood-clotting related problem			WGS, SNP genotyping	RNA-Seq, Microarray
Long term storage			human plasma metabolome is adequately stable to long-term storage at −80 °C for up to 7 y but significant changes occur upon longer storage Antje Wagner-Golbs 2019 [16].	Cryopreserved PBMCs have been reported to be stored up to 1.5 y, in a mechanical −70 °C/−80 °C freezer, depending on container type used, without effecting viable cell recovery [16].	you can storage whole blood at +4°C for 21 days using a CPD (Citrate, Phosphate, Dextrose) anti-coagulant solution They must be isolated from freshly

Uncertain Spans

location	transcription	uncertainty
ELAVL2 table / IP data tau cell	p-Tau (도 10)	(도 10) may be a reference marker (Fig 10) rendered in Korean; preserved verbatim.
ELAVL2 table / IP data APP cell	Ip p 34,35(APP)	”Ip p” reads as written; could be “Ip” with page reference p 34,35; preserved verbatim.
Blood top table / row labels	Innate (non-specific) / Adaptive (specific)	row labels are partly clipped on the left in fine crops; reading restored from full body but partial.
Long term storage / Whole blood cell	trailing bullet They must be isolated from freshly	sentence is clipped at the bottom edge of the photo.